NZ786536A - Methods of treating diseases associated with ILC2 cells - Google Patents

Abstract

Provided herein are compositions including compounds and/or cells for treating a disease associated with Group 2 innate lymphoid cells (ILC2s), and methods of treatment.

Description

METHODS OF TREATING DISEASES ATED WITH ILC2 CELLS The present application is a divisional application of New Zealand Application No. 754549, which is orated in its entirety herein by reference.

Group 2 innate lymphoid cells ) are abundant at mucosal rs and act as key initiators of type 2 inflammation and tissue repair1,2. ILC2s are activated by cell-extrinsic cytokines, including IL-25, IL-33 and thymic stromal lymphopoietin1,2. Previous reports indicated that discrete lymphocyte subsets and haematopoietic progenitors are controlled by dietary signals and neuroregulators2,3,5-9, suggesting that ILC2s may exert their function in the t of neuro-immune cell units.

SUMMARY As shown herein, the neuropeptide Neuromedin U has been determined to be a uniquely potent regulator of type 2 innate immunity in the context of a novel neuron-ILC2 unit. More specifically, it was determined that ILC2s express the Neuromedin U receptor 1 (Nmur1) while Neuromedin U is expressed by c neurons. Activation of ILC2s with Neuromedin U ed in prompt and strong production of the type 2 cytokines interleukin 5 (IL-5), IL-13 and Amphiregulin in a NMUR1-dependent manner. Neuromedin U controlled ILC2 downstream of ERK activation and calcium-influx-dependent activation of Calcineurin cytokines and NFAT.

Moreover, Neuromedin U treatment in vivo resulted in ate type 2 responses. Accordingly, ablation of Nmur1 led to impaired type 2 responses and poor worm infection control. Strikingly, mucosal neurons were found adjacent to ILC2s and directly sensed worm products to control Neuromedin U expression and innate type 2 cytokines. This work reveals novel neuro-immune interactions at the core of mucosal homeostasis indicating that neuron-ILC2 cell units are poised to confer ate tion via coordinated neuro-immune y responses.

According to one aspect, methods for increasing activity or proliferation of Group 2 innate lymphoid cells (ILC2s) are provided. The methods include contacting ILC2s with an agonist of edin U receptor 1 (NMUR1) in an amount effective to increase ty of the ILC2s. In some embodiments, the agonist of NMUR1 is neuromedin U (NMU) or an analog thereof, or an antibody that specifically binds and activates NMUR1 or an n-binding fragment thereof. In some embodiments, the NMU or analog thereof is NMU25, NMU precursor protein, NMU23, or NMU8.

In some embodiments, the contacting is in vitro. In some embodiments, the ILC2s are contacted in an ILC2 expansion protocol.

In other embodiments, the contacting is in vivo. In some embodiments, the agonist of neuromedin U receptor 1 (NMURl) is administered to a subject. In some embodiments, the subject is a human. In some embodiments, the subject is not ise in need of ent with the agonist of NMURl.

According to another aspect, s for treating a disease associated with Group 2 innate lymphoid cells (ILC2s) are provided. In some ments, the methods include administering to a t in need of such treatment an agonist of neuromedin U receptor 1 (NMURl) in an amount effective to treat the disease. In some embodiments, the agonist of NMURl is edin U (NMU) or an analog f, or an antibody that specifically binds and activates NMURl or an antigen—binding fragment thereof. In some embodiments, the NMU or analog thereof is NMU25, NMU precursor protein, NMU23, or NMU8. In some embodiments, the subject is a human.

In some embodiments, the disease is infection, tissue repair, wound healing, obesity, ble by increasing induction of type 2 immune responses, treatable by metabolic regulation, treatable by increasing phils, or treatable by increasing mast cells. In some embodiments, the subject is not otherwise in need of treatment with the agonist of NMURl.

In some embodiments, the agonist of NMURl is stered intravenously, orally, nasally, rectally or through skin absorption.

According to another aspect, agonists of neuromedin U receptor 1 (NMURl) are provided for use in treating a disease ated with Group 2 innate lymphoid cells (ILC2s) including administering to a subject in need of such ent the agonist of NMURl in an amount effective to treat the disease. In some embodiments, the agonist of NMURl is neuromedin U (NMU) or an analog thereof, or an antibody that specifically binds and activates NMURl or an antigen—binding fragment thereof. In some embodiments, the NMU or analog thereof is NMU25, NMU precursor protein, NMU23, or NMU8. In some ments, the t is a human.

In some embodiments, the disease is infection, tissue repair, wound healing, obesity, treatable by increasing induction of type 2 immune responses, treatable by metabolic regulation, treatable by increasing eosinophils, or treatable by increasing mast cells. In some embodiments, the subject is not otherwise in need of treatment with the agonist of NMURl.

In some embodiments, the agonist of NMURl is administered enously, orally, nasally, rectally or through skin tion.

According to another aspect, methods for treating a disease associated with Group 2 innate lymphoid cells (ILC2s) are provided. The methods include administering to a subject in need of such treatment a composition comprising activated ILC2s in an amount effective to treat the disease. In some embodiments, the composition further comprises an agonist of neuromedin U receptor 1 (NMURl). In some embodiments, the agonist of NMURl is neuromedin U (NMU) or an analog thereof, or an dy that specifically binds and tes NMURl or an antigen—binding fragment thereof. In some embodiments, the NMU or analog thereof is NMU25, NMU precursor protein, NMU23, or NMU8. In some embodiments, the subject is a human.

In some embodiments, the disease is infection, tissue repair, wound healing, obesity, treatable by increasing induction of type 2 immune ses, treatable by metabolic regulation, treatable by sing eosinophils, or treatable by sing mast cells. In some ments, the subject is not otherwise in need of treatment with the activated ILC2s or the agonist of NMURl.

In some embodiments, the ted ILC2s or the agonist of NMURl is administered enously, orally, nasally, rectally or through skin absorption.

According to another aspect, compositions are provided that include activated Group 2 innate lymphoid cells (ILC2s) for use in treating a disease associated with ILC2s including administering to a subject in need of such treatment the composition comprising ted ILC2s in an amount effective to treat the disease. In some embodiments, the composition further comprises an agonist of neuromedin U or 1 (NMURl). In some embodiments, the agonist of NMURl is neuromedin U (NMU) or an analog thereof, or an antibody that specifically binds and activates NMURl or an antigen—binding fragment thereof. In some embodiments, the NMU or analog thereof is NMU25, NMU precursor n, NMU23, or NMUS. In some embodiments, the subject is a human.

In some ments, the disease is infection, tissue repair, wound healing, obesity, treatable by increasing induction of type 2 immune responses, treatable by lic regulation, treatable by increasing eosinophils, or treatable by increasing mast cells. In some embodiments, the subject is not otherwise in need of treatment with the activated ILC2s or the agonist of NMURl.

In some embodiments, the activated ILC2s or the activated ILC2s and the agonist of NMURl is administered intravenously, orally, y, rectally or through skin tion.

According to another aspect, methods for decreasing activity or proliferation of Group 2 innate id cells (ILC2s) are provided. The methods include contacting ILC2s with an antagonist of neuromedin U receptor 1 (NMURl) or neuromedin U (NMU) in an amount effective to decrease activity of the ILC2s. In some embodiments, the antagonist of NMURl or NMU is an antibody that specifically binds and inhibits NMURl or NMU, respectively, or an antigen—binding fragment thereof. In some embodiments, the antagonist of NMURl or NMU is an inhibitory nucleic acid molecule that reduces that reduces sion, transcription or translation of NMURl or NMU. In some embodiments, the inhibitory nucleic acid is a sRNA, shRNA, or antisense nucleic acid molecule.

In some embodiments, the contacting is in vitro.

In other embodiments, the contacting is in vivo. In some embodiments, the antagonist of NMURl or NMU is administered to a subject. In some embodiments, the t is a human. In some ments, the subject is not otherwise in need of treatment with the antagonist of NMUR or NMU 1. ing to another aspect, methods for treating a disease associated with Group 2 innate lymphoid cells (ILC2s) are provided. The methods include stering to a subject in need of such treatment an antagonist of neuromedin U receptor 1 (NMURl) or neuromedin U (NMU) in an amount effective to treat the disease. In some embodiments, the antagonist of NMURl or NMU is an antibody that specifically binds and ts NMURl or NMU, respectively, or an antigen—binding fragment thereof. In some ments, the antagonist of NMURl or NMU is an tory nucleic acid molecule that reduces that reduces expression, transcription or translation of NMURl or NMU. In some embodiments, the inhibitory nucleic acid is a sRNA, shRNA, or antisense c acid molecule. In some embodiments, the subject is a human.

In some embodiments, the disease is allergy, ic asthma, food allergy, eosinophilic esophagitis, atopic dermatitis, fibrosis, allergic rhinitis, allergic rhinosinusitis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, treatable by reducing type 2 immune responses, treatable by reducing eosinophils, or treatable by reducing mast cells. In some embodiments, the subject is not otherwise in need of treatment with the agonist of NMURl or NMU.

In some embodiments, the antagonist of NMURl is administered intravenously, orally, nasally, rectally or through skin absorption. ing to another aspect, antagonists of neuromedin U receptor 1 (NMURl) or neuromedin U (NMU) are provided for use in treating a disease associated with Group 2 innate lymphoid cells (ILC2s) comprising administering to a subject in need of such treatment the nist of NMURl or NMU in an amount effective to treat the disease. In some embodiments, the antagonist of NMURl or NMU is an antibody that specifically binds and inhibits NMURl or NMU, tively, or an antigen—binding fragment thereof. In some embodiments, the antagonist of NMURl or NMU is an inhibitory nucleic acid molecule that reduces that reduces expression, transcription or translation of NMURl or NMU. In some embodiments, the inhibitory nucleic acid is a sRNA, shRNA, or antisense c acid molecule. In some embodiments, the subject is a human.

In some embodiments, the disease is allergy, allergic asthma, food allergy, philic esophagitis, atopic dermatitis, fibrosis, allergic rhinitis, allergic rhinosinusitis, chronic ctive ary disease (COPD), cystic fibrosis, treatable by reducing type 2 immune responses, treatable by reducing eosinophils, or treatable by reducing mast cells. In some embodiments, the subject is not otherwise in need of treatment with the t of NMURl or NMU.

In some embodiments, the antagonist of NMURl or NMU is administered intravenously, orally, nasally, rectally or through skin absorption.

The invention is not limited in its application to the s of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other ments and of being practiced or of being carried out in s ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having,:9 6Lcontaining,:9 6"involving," and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

BRIEF DESCRIPTION OF DRAWINGS The accompanying gs are not ed to be drawn to scale. In the drawings, each identical or nearly cal component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings: Figures 1a-1e. ILC2s express neuromedin U receptor 1 and closely locate with Neuromedin U-expressing neurons. Fig. 1a, Heat map for 40 neuronal—related mRNA transcripts in CD4 T cells, ILCls, ILC2s, NCR' (CD4+ and CD4") and NCR+ ILC3s subsetle.

Fig. 1b, Comparison of ILC2 gene expression with ILCl, ILC3 NCR+ and CD4 T cellle, by volcano plots. Nmur] is highlighted in red. Fig. 1c, Nmur] quantitative RT—PCR analysis in intestinal lamina propria cells unless stated otherwise. Common lymphoid progenitor (CLP); Common helper innate lymphoid progenitor (CHILP); Bone marrow ILC2 progenitor ); Eosinophils (Eo); Mast cells (Mast); Macrophages (Mo); Neutrophils (Neu); Dendritic cells (DC); T cells (T); B cells (B); Lamina propria glial cells (G) and neurons (N); Epithelial cells (Ep). n=6. Fig. 1d, Nmu quantitative RT—PCR is in intestinal populations. n=6. Fig. 1e, al analysis of intestinal lamina propria. Green: neurons (RetGFP); Red: KLRGl; Cyan: CD3. Cyan arrows: T cells (CD3+). Red : ILC2s.

Figures 2a-2j. Neuromedin U is a uniquely potent tor of innate type 2 cytokines, via NMURl activation. Figs. 2a-2f, ILC2—intrinsic activation with NmU23. Fig. 2a, Type 2 cytokine gene expression in intestinal ILC2s. n=6. Fig. 2b, Type 2 cytokine gene expression in lung ILC2s. n=6. Fig. 2c, Ki67 expression in intestinal ILC2s. Fig. 2d, IL—5 and IL— 13 expression in Nmur] competent and ent ILC2s. Fig. 2e, Innate inflammatory type 2 cytokines at the protein level. n=6. Fig. 2f, Innate —repair cytokine AREG. n=6. Figs. 2g,2h, in viva administration of NmU23. Fig. 2g, ILC2—derived type 2 cytokines. n=6. Fig. 2h, T erived type 2 cytokines. n=6. Figs. 2i,2j, in viva ablation of Nmur]. Fig. 2i, Intestinal ILC2s from Nmurl'l' and their Nmurl’"+ WT littermate controls. WT n=6; Nmurfl' n=9. Fig. 2j, ILC2—derived type 2 nes in bone marrow chimeras of Nmurfl' and in their Nmurl’"+ WT littermate control origin. WT n=6; Nmurfl' n=3. Error bars show s.e.m. >"P<0.05; >"*P"**P"***P Figures 3a-3e. Neuromedin U tes ILC2-derived cytokines via ERK1/2 and a Ca2+/Calcineurin/NFAT e. Figs. 3a-e, Intestinal ILC2 tion by Neuromedin U.

Fig. 3a, Percentage of pERK cells n=4. Mean fluorescence intensity (MFI) of pERK expression. n=4. Fig. 3b, 115, [[13 and Csz expression in ILC2s cultured with medium (control) (n=3), NmU23 (n=3) or NmU23 and ERK inhibitor PD98059 (n=3). Fig. 3c, Left and centre: Ca2+ influx, represented by Fluo—4 AM intensity. NmU23 was added 60 seconds after ILC2 ne acquisition (arrow). Right: Mean intensity of Ca2+ influx. n=3. Fig. 3d, 115, [[13 and Csz sion in ILC2s cultured with medium (control) (n=6), NmU23 (n=6) or NmU23 and Calcineurin inhibitor FK506 (n=6). Fig. 3e, [[5, [[13 and Csz expression in ILC2s cultured with medium (control) (n=3), NmU23 (n=3) or NmU23 and NFAT inhibitor llR—VIVIT (VIVIT) (n=3). Error bars show s.e.m. >"P"*P"***P Figures 4a-4h. The neuroregulatory axis NmU-NMURI confers protection against worm infection. Mice were infected with N. brasiliensis larvae and lungs analysed at 48 hours. Fig. 4a, Nmu sion in total lung from infected mice compared to non—infected controls. n=3. Fig. 4b, Pulmonary inflammatory cell infiltrates 48 hours after infection.

NmU23 treated and control sections are displayed. Hematoxylin and eosin. Fig. 4c, Myeloperoxidase— (granulocytes) and Luna—stained (eosinophils) lung sections. Fig. 4d, Granulocyte and eosinophil cell counts (cells/mmz). Control n=8; NmU23 n=8. Fig. 4e, Nmurfl' and their WT littermate controls were infected with N. brasiliensis. Hematoxylin and eosin. Fig. 4f, Myeloperoxidase— (granulocytes) and Luna—stained (eosinophils) lung sections. Fig. 4g, Granulocyte and eosinophilic cell counts (cells/mmz). WT n=8; Nmurfl' n=8. Fig. 4h, N. brasiliensis infection burden at 48 hours in the lung. WT n=3; Nmurl'l' n=3.

Scale bars: SOum. Error bars show s.e.m. >"P"*P"**P"***P Figures 6a-6f. Neuromedin U is potent regulator of lung innate type 2 cytokines, via NMURl activation. Figs. 6a,6b, ILC2—intrinsic activation with NmU23. Fig. 6a, IL—5 and IL—l3 expression in lung ILC2s. Fig. 6b, Innate type 2 cytokines at the protein level. n=3.

Figs. 6c,6d, in viva administration of NmU23. Fig. 6c, ILC2—derived type 2 nes in the lung. n=3. Fig. 6d, T cell—derived type 2 cytokines in the lung. n=3. Figs. 6e,6f, in viva ablation of Nmur]. Fig. 6e, Lung ILC2s in Nmurfl' and in their "+ WT littermate controls. WT n=6; Nmurfl' n=9. Fig. 6f, Intestinal T cell—derived type 2 cytokines in Nmurfl' and in their "+ WT littermate controls. WT n=6; Nmurl'l' n=6. Error bars show s.e.m. >"P<0.05; >"*P"**P"***P Figures 7a-7c. Nmurl is dispensable for ILC2 development. Figs. 7a,7c, Competitive bone marrow chimeras. Fig. 7a, 106 cells of each genotype (CD45.2) were injected enously in direct competition with a third—party WT competitor (CD45.l/CD45.2), in a 1:1 ratio, into non—lethally irradiated (150 Rad) NSG mice (CD45. 1).

Fig. 7b, Percentage and number of donor ILC2s in the intestine. WT n=l2; Nmurl'l' n=l2.

Fig. 7c, Percentage and number of donor ILC2s in the lung. WT n=l2; Nmurfl' n=l2. Error bars show s.e.m. >"P"*P"***P Figure 8. A novel neuron-ILC2 unit orchestrated by neuromedin U. Neuron— derived Neuromedin U ly activates ILC2s in a NMURl ent manner, resulting in a potent production of inflammatory and tissue repair type 2 cytokines that confer protection to worm infection. Neuromedin U activates NMURl with induces type 2 cytokine expression downstream of ERK phosphorylation and activation of a Ca2+/Calcineurin/NFAT cascade.

This model suggests that neuron—ILC2 cell units are poised to uniquely ensure potent and immediate type 2 responses in a neuromedin U—dependent manner.

Figures 9a-9i: Fig. 9a, Nmur] tative RT—PCR analysis in the lungs at day 6 post Nippostrongylus brasiliensis (NB) — infection in lung. Eosinophils (Eo); Mast cells (Mast); hages (Mo); phils (Neu); naive T cells (T); Innate lymphoid cells type 2 . Fig. 9b, NMUR] expression in human adaptive (CD4 T cells) and innate type 2 lymphocytes ILC2 from blood. Fig9c, Type 2 cytokine gene expression in human ILC2 and Th2 after in vitro stimulation with the peptide NmU25. Fig. 9d, Nmur] expression in lung ILC2 before and after infection (at day 6). Fig. 9e, Nmur] expression in steady state in Common lymphoid progenitor (CLP); Common helper innate lymphoid progenitor (CHILP), Bone marrow ILC2 progenitor (ILC2P) and E0, Mast, Mo, Neu, Dendritic cells (DC); naive T cells (T); T—helper 2 cells (Th2); memory T cells, B cells (B), Lamina Propria glial cells (G) and neurons (N). n=3—6. Fig. 9f, Type 2 cytokine gene expression in intestinal ILC2 and Th2 after in vitro stimulation with the peptide NmU23 (lOOng/mL). n=3—6. Fig. 9g, Confocal analysis of intestinal lamina propria. Green: neurons (RetGFP); Cyan: KLRGl; red: CD3.

Cyan: KLRGl. , Neurosphere—derived s. Red: TUJ 1. Blue: DAPI. Fig.9i, Activation of neurosphere—derived neurons with ns, TLR—ligands and N. brasiliensis excretory/secretory proteins (NES). >"P"**P"***P Figures 103-10d: Fig. 10a, Ki67 expression in intestinal ILC2s after an overnight in vitro stimulation with NmU23 alone (lOOng/mL, Phoenix Pharmaceutical) or NmU23 together with the survival cytokines Interleukin (IL)—2 and/or IL—7 (lOng/mL). Fig. 10b, Ki67 expression in ine ILC2 after in viva administration of NmU23 (4ug/day during 2 days). n=5. Fig. 10c, ILC2—derived type 2 cytokines (IL—5, IL—l3 and Amphiregulin (Areg)) in sorted intestine ILC2 after an overnight stimulation with NmU23, mouse recombinant IL—25 or IL—33 (R&D) (10, 50 and lOOng/mL). Negative control: unstimulated ILC2, Positive control: ILC2 activated with phorbol l2—myristate l3—acetate (PMA, 50ng/ml) plus ionomycin (500ng/ml). n=3. Fig. 10d, Dot plots representative of the cytokine production with increasing dose of NmU23, rIL—25 and . 05; .Ol; >"**P"***P Figures Ila-11b: ILC2 were FBS deprived for 2 hours prior to treatment with either (Fig. lla) llR—VIVIT (inhibits NFAT activation) (10 uM) or (Fig. 11b) cyclosporin A (CsA, lOOuM). Expression of type 2 cytokines were ed by quantitative RT—PCR (Figs. 11a,11b). n=3—6. Fig. 11c, Deprivated ILC2 from Lamina a were stimulated 90' with NmU23 (lOOng/mL), fixed, permeabilized and stained with anti—NFAT2 monoclonal antibody (abcam). Cells were analyzed by confocal microscopy. >"P"*P"*"‘P"***P Figures 123-12f: (Figs. c) Mice were infected with N. iensis larvae and treated with NmU23 peptide (Sug/day) or PBS (control). Lungs were analysed at day 2 post— infection. Fig. 12a, ILC2 response in lungs from NmU23 treated mice (n=5) compared to control (n=5). Fig. 12b, Burden of infection in lungs of infected mice treated with PBS (n=5) or NmU23 (n=5). Fig. 12c, Pulmonary hemorrhage in lung of infected mice treated with NmU23 compared to l. (Figs. 12d-12f) Mice were infected with N. brasiliensis larvae and treated with NmU23 peptide (Sug/day) or PBS (control). Lungs and small ine were analysed at day 6 post—infection. Fig. 12d, Neutrophils and eosinophils rate in broncho— alveolar lavage (BAL) in infected mice treated with NmU23 versus PBS. Control n=5; NmU23 n=5. Fig. 12e, Mastocytes and hages infiltrate in broncho—alveolar lavage (BAL) in infected mice treated with NmU23 versus PBS. Control n=5; NmU23 n=5. Fig. 12f, Burden of infection in small intestine of infected mice treated with PBS (n=5) or NmU23 (n25). Error bars show s.e.m. >"P"*P"**P"***P Figures 13a-13c: Nmurfl' and their WT mate controls were infected with N. iensis and analyzed at day 6 post—infection. Fig. 13a, ILC2 response in lungs of infected Nmurfl' and their WT littermate controls D6 post—infection. WT n=6; Nmurl'l' n=8. Fig. 13b, Neutrophils (Neu) and eosinophils (Eos) infiltrate in broncho—alveolar lavage (BAL) in infected Nmurfl' and their WT littermate controls. WT n=6; l' n=7. Fig. 13c, Mastocytes and hages infiltrate in o—alveolar lavage (BAL) in infected l' and their WT littermate controls. WT n=6; Nmurl'l' n=7. Error bars show s.e.m. >"P"*P"**P"***P Figures 14a-14c: Competitive bone marrow chimeras treated with NmU23. Fig. 14a, 106 cells of each genotype (CD452) were ed intravenously in direct competition with a third—party WT competitor (CD45. l/CD45.2), in a 1:1 ratio, into non—lethally irradiated (150 Rad) NSG mice (CD451). The mice received one injection of PBS or NmU23 (20pg). Fig. 14b, Percentage and number of donor ILC2s in the lungs. WT n=5; Nmurfl' n=5. Fig. 14c, Percentage and number of donor T cells in the lungs. WT n=5; Nmurl'l' n=5. Error bars show s.e.m. >"P"*P"**P"***P DETAILED DESCRIPTION Group 2 innate id cells (ILC2s) are major regulators of inflammation, tissue repair and metabolic homeostasisl’z. ILC2 activation has been shown by host—derived nes and alarminsl’z, but, how ILC2s respond to neuronal—derived signals remains unclear.

As described herein, it was determined that ILC2s express the Neuromedin U receptor 1 (Nmurl ) and that the neuropeptide Neuromedin U is a potent activator of ILC2s.

Neuromedin U resulted in prompt and strong production of the type 2 nes interleukin 5 (IL—5), IL—l3 and Amphiregulin in a NMURl—dependent manner. Neuromedin U controlled ILC2 downstream of ERK activation and calcium—influX—dependent tion of Calcineurin cytokines and NFAT. When used in viva, Neuromedin U treatment resulted in immediate type 2 responses. It also was shown that ablation of Nmur] led to impaired type 2 responses and poor worm infection control.

Increasing activity of ILC2s The s sed herein include methods for sing activity or proliferation of Group 2 innate lymphoid cells ) by contacting ILC2s with an agonist of neuromedin U receptor 1 (NMURl) in an amount effective to increase activity of the ILC2s.

The methods disclosed herein also include s for treating a disease associated with Group 2 innate lymphoid cells (ILC2s) by administering to a subject in need of such treatment an agonist of neuromedin U receptor 1 (NMURl) in an amount effective to treat the disease.

Other methods for treating e include administering to a subject in need of such ent a composition comprising activated ILC2s in an amount effective to treat the disease. In some of these methods, the ition comprising activated ILC2s also includes an agonist of neuromedin U receptor 1 (NMURl). Alternatively, an agonist of NMURl can be administered tely from the composition comprising ted ILC2s.

Also provided herein are agonists of NMURl for use in treating a disease associated with ILC2s, and compositions comprising ILC2s (and optionally an agonist of NMURl) for use in treating a disease associated with ILC2s.

As used herein, neuromedin U receptor 1 (NMURl) is a 7 transmembrane receptor of the rhodopsin , and is also known as FM3, FM—3, GPC—R, G—protein coupled receptor 66 (GPR66), and NMUlR. As described elsewhere herein, an agonist of NMURl includes a neuromedin U (NMU) or an analog thereof, an antibody that specifically binds and activates NMURl or an antigen—binding fragment thereof, or a small molecule ligand of NMURl.

Contacting ILC2s with an agonist of NMURl can be performed in vitro, such as in an ILC2 expansion protocol performed to produce ILC2s, or can be performed in vivo. In some embodiments of s in which the contacting of ILC2s with an agonist of NMURl is performed in vivo, the agonist of NMURl is administered to a subject, such as a human. In some of these s, the t is not otherwise in need of treatment with the agonist of NMURl.

In the disclosed methods, the subject can be a human. In some of these methods, the subject is not otherwise in need of treatment with the agonist of NMURl and/or treatment with the activated ILC2s.

Diseases treatable by the disclosed methods include infection, tissue repair, wound healing, obesity, diseases treatable by increasing ion of type 2 immune responses, WO 09540 diseases treatable by metabolic regulation, diseases treatable by increasing eosinophils, and diseases treatable by increasing mast cells.

The agonist of NMURl and/or the activated ILC2s can be administered by any suitable route of administration or delivery method. Suitable routes of administration e intravenous, oral, nasal, rectal or through skin absorption.

The agonist of NMURl and/or the activated ILC2s can be administered at any suitable interval, including daily, twice daily, three times per day, four times per day, every other day, weekly, every two weeks, every four weeks, continuously (e.g., by infusion, patch, or pump), and so on.

Decreasing activity of ILC2s Additional methods disclosed herein include methods for decreasing activity or eration of Group 2 innate id cells (ILC2s) by contacting ILC2s with an antagonist of neuromedin U receptor 1 (NMURl) or an nist of NMU (or both) in an amount effective to decrease ty of the ILC2s.

The methods disclosed herein also include methods for treating a e associated with Group 2 innate lymphoid cells (ILC2s) by administering to a subject in need of such treatment an antagonist of neuromedin U receptor 1 (NMURl) in an amount ive to treat the disease.

Also provided herein are antagonists of NMURl for use in treating a disease associated with ILC2s.

As described elsewhere herein, an antagonist of NMURl includes an inhibitory nucleic acid molecule that reduces that reduces expression, transcription or translation of NMURl, such as a sRNA, shRNA, or antisense nucleic acid molecule; an antibody that specifically binds and inhibits NMURl or an antigen—binding fragment thereof, or a small le antagonist of NMURl.

Contacting ILC2s with an nist of NMURl can be performed in vitro, or can be med in vivo. In some embodiments of methods in which the ting of ILC2s with an antagonist of NMURl is performed in vivo, the antagonist of NMURl is administered to a subject, such as a human. In some of these methods, the subject is not otherwise in need of treatment with the antagonist of NMURl.

In the disclosed methods, the subject can be a human. In some of these methods, the subject is not otherwise in need of ent with the antagonist of NMURl.

In the methods disclosed herein for treating disease by administering an nist of NMURl, the disease can be allergy, allergic asthma, food allergy, eosinophilic esophagitis, atopic dermatitis, fibrosis, allergic rhinitis, allergic inusitis, c ctive pulmonary disease , cystic is, diseases treatable by reducing type 2 immune responses, diseases treatable by reducing phils, or diseases treatable by reducing mast cells.

The nist of NMURl can be administered by any suitable route of administration or delivery method. Suitable routes of administration include intravenous, oral, nasal, rectal or through skin absorption.

The antagonist of NMURl can be administered at any suitable interval, including daily, twice daily, three times per day, four times per day, every other day, weekly, every two weeks, every four weeks, continuously (e.g., by infusion, patch, or pump), and so on.

Agonists of neuromedin U receptor 1 gNMURl) Agonists of NMURl include peptide agonists (including modified es and conjugates), activating antibody molecules, and small molecules. Peptide agonists include neuromedin U (also known as and referred to herein as NMU or NmU) or analogs thereof.

The NMURl agonists may be entirely specific for NMURl, may agonize NMURl preferentially (as compared to edin U receptor 2, NMUR2), or may agonize both NMURl and NMUR2. Such agonists may be useful even if NMURl is agonized less than NMUR2, but it is preferred that the ts used in the methods described herein agonize NMURl to a greater extent than NMUR2. As used herein agonizing NMURl preferentially (as compared to neuromedin U receptor 2, NMUR2) means that the agonist agonizes NMURl at least 10%, 25%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more than NMUR2.

Neuromedin U (also referred to herein as NMU) is a neuropeptide conserved in many species, which was isolated as a peptide consisting of 25 amino acid residues (NMU—25) or as a peptide consisting of 8 amino acid residues (NMU—8), from pig small intestine. NMU—8 consists of the C—terminal 8 residues of porcine NMU25. NMU—25 also is present in humans, and is preferred for use in humans. The C—terminal 8 amino acid residues of human NMU—25 (also referred to as NMU—8) are the same as that of the C—terminal 8 amino acid residues of porcine NMU—8. The 8 amino acids at the C terminus of NMU—25 are the most highly conserved and this peptide has been shown to have similar activity as . Rat NMU consists of 23 amino acid es, and is known as NMU—23. The amino acid sequence of the C—terminal 8 residues of rat NMU—23 differs from that of the inal 8 residues of porcine NMU—8 by one amino acid residue. NMU precursor protein (and its cleaved peptides) also can be used in the methods described herein. NMU precursor protein is a 174 amino acid long protein.

Amino acid sequences of the NMU sor protein and NMU are provided as follows: NMU precursor protein (P48645INMU_HUMAN Neuromedin—U OS=Homo sapiens GN=NMU PE=l SV=l) MLRTESCRPRSPAGQVAAASPLLLLLLLLAWCAGACRGAPILPQGLQPEQQLQLWNE IDDTCSSFLSIDSQPQASNALEELCFMIMGMLPKPQEQDEKDNTKRFLFHYSKTQKLG KSNVVSSVVHPLLQLVPHLHERRMKRFRVDEEFQSPFASQSRGYFLFRPRNGRRSAG FI (SEQ ID NO: 1) NMU25 FRVDEEFQSPFASQSRGYFLFRPRN (SEQ ID NO: 2) NMU23 FKAEYQSPSVGQSKGYFLFRPRN (SEQ ID NO: 3) NMU8 RN (SEQ ID NO: 4) Agonists of NMURl include NMU analogs, derivatives, and conjugates, such as NMU analogs having variations in amino acid sequence relative to natural NMU sequences but which retain function of binding to and activating NMURl. Other examples of analogs, tives, and ates of NMU include: the modified peptides of Takayama et al. (ACS Med Chem Lett. 2015 Mar 12; 6(3): 302—307); the NMU—8 analogs of Inooka et al. (Bioorg Med Chem. 2017 Feb 21. pii: S0968—0896(17)30108—6); the PEGylated derivatives of NMU of Ingallinella et al. (Bioorg Med Chem. 2012 Aug l;20(15):4751—9); the human serum albumin (HSA)—NMU conjugate of Neuner et al. (J Pept Sci. 2014 Jan;20(l):7—l9); the truncated/lipid—conjugated NMU analogs of Micewicz (Eur J Med Chem. 2015 Aug 28;101:6l6—26); and the lipidated NMU analogs of Dalboge et al. (J Pept Sci. 2015 (2):85-94).

As described in US 2011/0294735 and by reference for the specific recitation of the following compounds), additional NMURl agonists comprise the general formula (I) Zl-peptide-ZZ (I) wherein the e has the amino acid sequence X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15 X16 X17 X18 X19 X20 X21 X22 X23 X24—X25, wherein amino acids 1 to 17 can be any amino acid or absent, n amino acid X18 is absent, Y, W, F, a des—amino acid or an acyl group; amino acid X19 is A, W, Y, F or an aliphatic amino acid; amino acid X20 is absent, L, G, sarcosine (Sar), D—Leu, NMe—Leu, D— Ala or A; amino acid X21 is F, NMe—Phe, an aliphatic amino acid, an aromatic amino acid, A or W; X22 is R, K, A or L; amino acid X23 is P, Sar, A or L; amino acid X24 is R, Harg or K; and amino acid X25 is N, any D— or L—amino acid, Nle or D—Nle, A; and Z1 is an optionally present protecting group that, if present, is joined to the N—terminal amino group; and Z2 is NH2 or an optionally present ting group that, if present, is joined to the C—terminal carboxy group, and pharmaceutically able salts thereof.

As described in US 2012/0094898 (incorporated herein by reference for the specific recitation of the following compounds), additional NMURl ts include peptide derivatives ed from the group consisting of (AL)—B—Ala—Tyr—Nal(l)—Leu—Phe—Arg—Pro—Arg—Asn—NH2, PEG20k(AL)—B—Ala—Tyr—Nal(2)—Leu—Phe—Arg—Pro—Arg—Asn—NH2, PEG20k(AL)—NpipAc—Tyr—Nal(2)—Leu—Phe—Arg—Pro—Arg—Asn—NH2, PEG20k(AL)—NpipAc—Tyr—Nal(2)—Leu—Phe—Arg—Ala—Arg—Asn—NH2.

PEG20k(AL)—PEG(2)—Tyr—Nal(2)—Leu—Phe—Arg—NMeAla—Arg—Asn—NH2, PEG20k(AL)—Pic(4)—Tyr—Nal(2)—Leu—Phe—Arg—NMeAla—Arg—Asn—NH2, PEG20k(AL)—Acp—Tyr—Nal(2)—Leu—Phe—Arg—NMeAla—Arg—Asn—NH2, and WO 09540 PEG20k(AL)—B—Ala—Tyr—Nal(2)—Leu—Pya(4)—Arg—Pro—Arg—Asn—NH2, or a salt of any of the peptide derivatives.

As described in recitation of the ing compounds), additional NMURl agonists e compositions sing the formula Zl—peptide—Z2 wherein the peptide has the amino acid sequence X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15 X16 X17 X18 X19 X20 X21 X22 X23 X24—X25, wherein amino acids 1 to 17 can be any amino acid or absent; wherein amino acid X18 is absent, Tyr or D—Tyr, Leu, Phe, Val, Gln, Nle, Glu or D—Glu, Asp, Ala, D—Lys, an aromatic amino acid, a ino acid or an acyl group; amino acid X19 is Ala, Trp, Tyr, Phe, Glu, Nva, Nle or an aromatic amino acid; amino acid X20 is absent, Leu, Gly, sarcosine (Sar), D—Leu, u, D—Ala or Ala, or any D— or L—amino acid; amino acid X21 is Phe, NMe— Phe, an aliphatic amino acid, an aromatic amino acid, Ala or Trp; X22 is Arg, Lys, Harg, Ala, or Leu; amino acid X23 is Pro, Ser, Sar, Ala or Leu; amino acid X24 is Arg, Harg or Lys; and amino acid X25 is Asn, any D— or L—amino acid, Nle or D—Nle, D—Ala or Ala; Z1 is optionally a protecting group that, if t, is joined to the N—terminus amino group; and Z2 is NH2 or an optionally t protecting group that, if present, is joined to the C—terminal carboxy group, and pharmaceutically acceptable salts thereof.

As described in recitation of the following compounds), additional NMURl agonists include compositions comprising a neuromedin U receptor agonist in which neuromedin U or an analog thereof is conjugated to cysteine residue 34 of human serum albumin by a non—maleimido or non— succinimidyl linkage or a pharmaceutically acceptable salt thereof.

As described in WC 42053 (incorporated herein by reference for the specific recitation of the following compounds), additional NMURl agonists include a neuromedin U or agonist represented by the following formula: Zl—peptide—Z2 wherein the peptide has the amino acid sequence ILQRGSGTAAVDFTKKDHTATWGRPFFLFRPRN (SEQ ID NO: 5), wherein the e can have one or more insertions or substitutions of the amino acid sequence with an alternative amino acid and wherein the peptide can have one or more deletions of the amino acid ce; Z1 is an optionally t protecting group that, if present, is joined to the N— al amino group; and Z2 is NH2 or an optionally present protecting group that, if present, is joined to the C—terminal carboxy group; and pharmaceutically acceptable salts thereof.

As bed in recitation of the following compounds), additional NMURl agonists include neuromedin U derivatives selected from polypeptides consisting of an amino acid sequence which is bound with a methoxypolyethylene glycol(s) via a linker, wherein the amino acid sequence contains at least 8 amino acids of the C—terminus of an amino acid sequence of neuromedin U, and is the same or ntially the same as the amino acid sequence of neuromedin U.

Antagonists of neuromedin U or 1 gNMURl ) or Neuromedin U (NMU) Antagonists of NMURl include peptide antagonists ding modified peptides and conjugates), inhibitory antibody molecules, inhibitory nucleic acid molecules, and small molecules. The NMURl antagonists may be entirely specific for NMURl, may antagonize NMURl preferentially (as compared to neuromedin U receptor 2, NMUR2), or may nize both NMURl and NMUR2. Such antagonists may be useful even if NMURl is antagonized less than NMUR2, but it is preferred that the antagonists used in the methods described herein antagonize NMURl to a r extent than NMUR2. As used herein, antagonizing NMURl preferentially (as compared to neuromedin U receptor 2, NMUR2) means that the antagonist antagonizes NMURl at least 10%, 25%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more than NMUR2.

As described in US 165144 (incorporated herein by reference for the specific recitation of the following compounds), additional NMU and NMURl antagonists include (i) a neuromedin U (NMU)—specific inhibitory nucleic acid, e.g., an siRNA, antisense, aptamer, or me targeted specifically to NMU; (ii) a neuromedin U (NMU) inhibitory peptide, e.g., a peptide comprising the sequence Phe—Arg—Pro—Arg—Asn (SEQ ID NO: 6); or (iii) an antibody or antigen binding nt thereof that binds to an NMU—R, e.g., NMU—R1, and inhibits NMU signalling, e.g., inhibits binding of NMU to the NMU—R1.

Suitable NMURl antagonists also can include: (i) a neuromedin U receptor 1 (NMURl)—specific inhibitory nucleic acid, e.g., an siRNA, antisense, aptamer, or ribozyme targeted specifically to NMURl; or Suitable NMU antagonists also can include: (i) a soluble NMURl molecule that binds NMU, such as an ellular portion of NMURl (e.g., amino acids 1 — 65 of UniProtKB — Q9HB89) optionally linked or fused to another polypeptide sequence for stability or other functions, such as an immunoglobulin Fc region; and (ii) an antibody or antigen binding fragment thereof that binds to an NMU, e.g., NMU—8, NMU—23, or NMU—25, and inhibits NMU ling, e.g., inhibits binding of NMU to the NMU—R1.

A subject shall mean a human or vertebrate mammal including but not limited to a dog, cat, horse, goat and non—human primate, e.g., monkey. ably the subject is a human. In some embodiments the subject is one who is not otherwise in need of treatment with an NMURl agonist or NMURl antagonist. Therefore the t, in specifically identified embodiments, may be one who has not been previously diagnosed with a disorder for which an NMURl agonist or NMURl antagonist is an identified form of ent.

The t can be first identified as a subject in need of ent, such as one having a disease that is treatable by the s disclosed herein, and then treated with an NMURl agonist (and/or activated ILC2s) or NMURl nist. The skilled artisan is aware of methods for identifying a subject as having a disease that is treatable by the methods disclosed herein.

As used , the terms "treat,:9 6‘treated," or "treating" refers to a treatment of a disease that ameliorates the disease (disease cation), rates symptoms of the disease, prevents the disease from ng worse, or slows the progression of the disease compared to in the absence of the therapy.

A "disease associated with Group 2 innate lymphoid cells (ILC2s)" as used herein is a disease or disorder in which ILC2s play some role in the development, maintenance or worsening of the disease or disorder.

In some of the methods disclosed herein, such diseases can be effectively treated by increasing ty or proliferation of ILC2s, such as by contacting ILC2s with an agonist of neuromedin U receptor 1 (NMURl) in an amount effective to increase activity of the ILC2s; by administering to a subject in need of such treatment an agonist of NMURl in an amount effective to treat the disease; or by stering activated ILC2s (and optionally an agonist of NMURl) in an amount ive to treat the e.

Diseases treatable by such methods include: infection, tissue repair, wound healing, obesity, diseases treatable by increasing induction of type 2 immune responses, diseases treatable by lic regulation, diseases treatable by increasing eosinophils, and diseases treatable by increasing mast cells In other of the method disclosed herein, the diseases can be effectively treated by decreasing activity or proliferation of ILC2s, such as by contacting ILC2s with an antagonist of neuromedin U receptor 1 (NMURl) in an amount effective to decrease activity of the ILC2s; or by administering to a subject in need of such treatment an antagonist of NMURl in an amount effective to treat the disease. es treatable by such methods include: allergy, allergic asthma, food allergy, philic esophagitis, atopic dermatitis, fibrosis, allergic rhinitis, allergic rhinosinusitis, chronic obstructive ary disease (COPD), cystic fibrosis, diseases treatable by reducing type 2 immune responses, diseases treatable by reducing eosinophils, or diseases treatable by reducing mast cells.

Toxicity and cy of the methods of the present invention can be determined by rd pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) or TD50 (the dose toxic to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be sed as the ratio Dso or D50. Therapeutic agents that exhibit large therapeutic indices are preferred. While therapeutic agents that exhibit toxic side effects may be used, in such cases it is preferred to use a ry system that targets such agents to the site of affected tissue in order to minimize potential damage to other cells or tissues and, thereby, reduce side effects.

The data obtained from the cell culture assays and/or animal studies can be used in formulating a range of dosage of the therapeutic agents for use in humans. The dosage of such agents lies preferably Within a range of circulating concentrations that include the ED50 with little or no ty. The dosage may vary Within this range depending upon the dosage form employed and the route of stration utilized. For any agent used in the method of WO 09540 the invention, the therapeutically effective dose can be estimated initially from cell culture . A dose may be ated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half—maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.

In certain embodiments, ceutical compositions may comprise, for example, at least about 0. l% of an active nd. In other embodiments, the an active compound may se between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for e, and any range derivable therein. Other, higher tages of an active compound also can be used.

The pharmaceutical compositions may also be, and preferably are, sterile in some ments. In other embodiments the compounds may be isolated. As used herein, the term "isolated" means that the nced material is removed from its native environment, e.g. a cell. Thus, an isolated biological material can be free of some or all cellular components, i.e., components of the cells in which the native material is occurs naturally (e. g., cytoplasmic or membrane components). In the case of nucleic acid molecules, an isolated nucleic acid includes a PCR product, an isolated RNA, a synthetically (e. g., chemically) produced RNA, such as an siRNA, an antisense nucleic acid, an aptamer, etc.

Isolated nucleic acid molecules include sequences inserted into plasmids, cosmids, or other vectors to form part of a chimeric recombinant nucleic acid construct, or produced by sion of a nucleic acid encoding it. Thus, in a specific embodiment, a inant nucleic acid is an isolated nucleic acid. An isolated n may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane—associated protein, or may be synthetically (e.g., chemically) produced, or produced by expression of a nucleic acid encoding it. An isolated cell, such as an ILC2 cell, can be removed from the anatomical site in which it is found in an organism, or may be produced by in vitro expansion of an isolated cell or cell population. An isolated material may be, but need not be, purified.

The term "purified" in reference to a protein, a nucleic acid, or a cell or cell population, refers to the separation of the d substance from contaminants to a degree ient to allow the practitioner to use the purified substance for the desired purpose.

Preferably this means at least one order of magnitude of purification is achieved, more ably two or three orders of magnitude, most preferably four or five orders of magnitude of purification of the starting material or of the natural material. In specific embodiments, a purified agonist of NMURl or antagonist of NMURl or ILC2 population is at least 60%, at least 80%, or at least 90% of total protein or nucleic acid or cell population, as the case may be, by weight. In a specific embodiment, a purified agonist of NMURl or antagonist of NMURl or ILC2 population is purified to homogeneity as assayed by standard, relevant laboratory protocols.

In some ments a purified and or isolated molecule is a synthetic molecule. t doses of the compounds described herein typically range from about 0.1 ug to 10,000 mg, more typically from about 1 ug/day to 8000 mg, and most typically from about ug to 100 ug. Stated in terms of subject body weight, typical dosages range from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body , about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 ram/kg/body weight, about 1 ram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 ram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable n. In non—limiting examples of a derivable range from the numbers listed herein, a range of about 1 mg/kg/body weight to about 100 mg/kg/body , about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above. The absolute amount will depend upon a variety of factors including the rent treatment, the number of doses and the individual patient parameters including age, physical ion, size and weight. These are factors well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment. Multiple doses of the molecules of the invention are also contemplated.

The nds and/or cells described herein may be used alone t other active therapeutics or may be combined with other therapeutic compounds for the treatment of the diseases described herein.

When used in combination with the compounds and cells described herein, the dosages of known therapies may be reduced in some instances, to avoid side effects. In some instances, when the compounds and/or cells bed herein are administered with r therapeutic, a sub—therapeutic dosage of either the compounds and/or cells described herein or the known therapies, or a sub—therapeutic dosage of both, is used in the treatment of a t.

A "sub—therapeutic dose" as used herein refers to a dosage which is less than that dosage which would produce a eutic result in the subject if administered in the absence of the other agent. Thus, the sub—therapeutic dose of a known therapy is one which would not produce the desired therapeutic result in the subject in the absence of the administration of the compounds and cells described herein. Existing therapies for the diseases bed herein are well known in the field of medicine, and may be described in references such as Remington’s Pharmaceutical Sciences; as well as many other l references relied upon by the medical profession as guidance for treatment.

When the compounds and/or cells described herein are administered in combination with other therapeutic agents, such administration may be simultaneous or sequential. When the other therapeutic agents are stered simultaneously they can be administered in the same or separate formulations, but are administered at the same time. The administration of the other therapeutic agent and the compounds and/or cells described herein can also be temporally separated, meaning that the other therapeutic agents are stered at a different time, either before or after, the administration of the compounds and cells described herein.

The separation in time between the administration of these compounds may be a matter of minutes or it may be longer.

The active agents of the invention (e.g., the compounds and cells described herein) are administered to the subject in an effective amount for treating disease. According to some aspects of the invention, an effective amount is that amount, depending on the disease being treated, of a NMURl t r activated ILC2s) or NMURl antagonist alone or in combination with another medicament, which when combined or co—administered or administered alone, results in a therapeutic response to the disease. The biological effect may be the amelioration and or absolute elimination of disease, or of symptoms ing from the disease. In another embodiment, the biological effect is the complete tion of the disease, as evidenced for e, by the absence of a symptom of the disease.

WO 09540 The effective amount of a compound (i.e., any of the agonists, antagonists, or ILC2s) used in s of the invention in the treatment of a disease described herein may vary depending upon the ic compound used, the mode of delivery of the compound, and whether it is used alone or in combination. The effective amount for any ular application can also vary depending on such factors as the disease being treated, the particular compound being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a ular molecule of the invention using routine and accepted methods known in the art, without necessitating undue experimentation. Combined with the teachings provided herein, by choosing among the various active compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side—effects and preferred mode of administration, an effective therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is effective to treat the particular subject.

Pharmaceutical itions of the present invention comprise an ive amount of one or more agents, dissolved or dispersed in a pharmaceutically acceptable r. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular es and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. er, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, l safety and purity standards as required by relevant government regulatory agencies. The nds are generally suitable for administration to humans.

This term requires that a compound or composition be nontoxic and sufficiently pure so that no further lation of the compound or composition is needed prior to administration to humans.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption ng agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences , incorporated herein by reference). Except r as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is plated.

The therapeutic compositions used as described herein may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and r it need to be sterile for such routes of administration as injection. The compounds and/or cells bed herein can be administered intravenously, intradermally, intraarterially, intralesionally, intracranially, intraarticularly, intranasally, intravitreally, intravaginally, intrarectally, topically, intramuscularly, intraperitoneally, subcutaneously, intravesicularlly, mucosally, orally, locally, by inhalation (e.g., aerosol inhalation), by ion, by infusion including by continuous infusion, by localized ion, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the foregoing as would be known to one of ordinary skill in the art (see, for example, ton's Pharmaceutical Sciences) and as is appropriate for the disease being d.

In any case, the composition may se various antioxidants to retard oxidation of one or more components. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal , including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

The compounds described herein may be formulated into a ition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, ic or mandelic acid. Salts formed with the free carboxyl groups also can be d from inorganic bases such as for example, sodium, potassium, um, m or ferric hydroxides; or such organic bases as isopropylamine, hylamine, histidine or procaine.

In embodiments where the compounds and/or cells described herein is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, eta), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example ypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or ations thereof.

The compounds and/or cells described herein can be administered in various ways and to different classes of recipients. In some instances the administration is chronic. Chronic administration refers to long term administration of a drug to treat a disease. The chronic administration may be on an as needed basis or it may be at regularly scheduled intervals.

For instance, the nds and/or cells described herein may be administered twice daily, three times per day, four times per day, every other day, weekly, every two weeks, every four weeks, continuously (e.g., by on, patch, or pump), and so on.

The compounds and/or cells described herein may be administered directly to a tissue.

Direct tissue administration may be achieved by direct injection. The compounds may be stered once, or alternatively they may be stered in a plurality of administrations.

If administered multiple times, the compounds may be administered via different routes. For example, the first (or the first few) administrations may be made directly into the affected tissue while later administrations may be systemic.

The compounds and/or cells described herein are administered in pharmaceutically acceptable solutions, which may ely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other eutic ingredients.

According to the methods described herein, the compounds and/or cells described herein may be administered in a pharmaceutical composition. In general, a pharmaceutical composition comprises the compound of the invention and a pharmaceutically—acceptable carrier. Pharmaceutically—acceptable carriers useful with compounds and/or cells described herein are well—known to those of ordinary skill in the art. As used herein, a pharmaceutically—acceptable carrier means a non—toxic material that does not interfere with the effectiveness of the biological activity of the compounds and/or cells described herein.

Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, lizers and other materials which are well—known in the art. Exemplary pharmaceutically acceptable rs for peptides in particular are bed in US. Patent No. 657. Such preparations may routinely n salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the WO 09540 salts should be pharmaceutically acceptable, but non—pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically—acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically— acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, lic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically—acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.

The compounds and/or cells described herein may be formulated into preparations in solid, semi—solid, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections, and usual ways for oral, parenteral or surgical administration. The ion also es pharmaceutical compositions which are ated for local administration, such as by implants.

Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, es, each containing a predetermined amount of the active agent.

Other compositions include suspensions in aqueous liquids or non—aqueous liquids, such as a syrup, an elixir or an emulsion.

For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as s, pills, dragees, capsules, liquids, gels, syrups, slurries, sions and the like, for oral ingestion by a t to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; ose preparations such as, for example, maize starch, wheat starch, rice starch, potato , n, gum tragacanth, methyl cellulose, hydroxypropylmethyl—cellulose, sodium ymethylcellulose, and/or nylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross—linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any carriers.

Dragee cores are provided with le coatings. For this purpose, concentrated sugar ons may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene , and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. ceutical preparations which can be used orally include push—fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push—fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as es, and/or lubricants such as talc or magnesium te and, ally, stabilizers. In soft capsules, the active compounds may be dissolved or ded in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. pheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.

For buccal administration, the itions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds and/or cells described herein may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered . Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. Techniques for preparing aerosol delivery systems are well known to those of skill in the art. Generally, such s should utilize components which will not significantly impair the biological properties of the active agent (see, for example, Remington’s Pharmaceutical Sciences). Those of skill in the art can readily ine the various parameters and conditions for producing aerosols without resort to undue experimentation.

The compounds, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. ations for injection may be presented in unit dosage form, e.g., in ampoules or in multi—dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or s vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Preparations for eral administration include e s or non—aqueous ons, suspensions, and emulsions. Examples of non—aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti—oxidants, chelating agents, and inert gases and the like. Lower doses will result from other forms of stration, such as intravenous administration. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance s. Multiple doses per day are contemplated to e appropriate ic levels of compounds.

In yet other embodiments, e for the compounds and/or cells described herein is a biocompatible microparticle or implant that is suitable for tation into a mammalian recipient. Exemplary bioerodible implants are known in the art. The implant may be a polymeric matrix in the form of a microparticle such as a microsphere (wherein the agent is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the agent is stored in the core of a polymeric shell). Other forms of the polymeric matrix for containing the agent include films, coatings, gels, implants, and stents. The size and composition of the ric matrix device is selected to result in favorable release cs in the tissue into which the matrix device is implanted. The size of the polymeric matrix device further is ed according to the method of ry which is to be used, typically injection into a tissue or administration of a suspension by aerosol into the nasal and/or pulmonary areas.

The polymeric matrix ition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when the device is administered to a vascular, pulmonary, or other surface. The matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time.

Both non—biodegradable and biodegradable polymeric matrices can be used to deliver the compounds and/or cells bed herein to the subject. Biodegradable matrices are preferred. Such polymers may be natural or synthetic polymers. The polymer is selected based on the period of time over which e is d, generally in the order of a few hours to a year or longer. lly, release over a period ranging from between a few hours and three to twelve months is most desirable. The polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, ally is linked with multivalent ions or other rs.

In general, the compounds and/or cells described herein may be delivered using the dible implant by way of diffusion, or more preferably, by degradation of the polymeric matrix. ary synthetic polymers which can be used to form the biodegradable ry system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly—vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co—polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose , cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, y—propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, ose propionate, cellulose acetate butyrate, ose acetate phthalate, carboxylethyl cellulose, cellulose tate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), inyl alcohols), polyvinyl acetate, poly vinyl chloride, polystyrene and polyvinylpyrrolidone.

Examples of non—biodegradable polymers include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

Other delivery systems can include time—release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compound, increasing convenience to the subject and the physician. Many types of e delivery WO 09540 systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide—glycolide), copolyoxalates, prolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Such delivery systems also include non—polymer systems such as lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono— di— and tri— glycerides; hydrogel release systems; silastic s; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. In addition, pump—based hardware delivery systems can be used, some of which are d for implantation.

Use of a long—term sustained release implant may be particularly suitable for treatment of chronic diseases. Long—term release, as used herein, means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 30 days, and preferably at least 60 days. Long—term sustained e implants are well—known to those of ordinary skill in the art and include some of the systems described above.

Thus the compounds and/or cells bed herein described herein may, in some embodiments, be assembled into pharmaceutical or research kits to facilitate their use in therapeutic or research applications. A kit may include one or more containers housing the components of the invention and ctions for use. Specifically, such kits may include one or more compounds and/or cells described herein, along with instructions describing the intended therapeutic application and the proper administration of these agents. In certain embodiments the compounds and/or cells described herein in a kit may be in a pharmaceutical ation and dosage suitable for a particular application and for a method of administration of the agents.

The kit may have a variety of forms, such as a blister pouch, a shrink wrapped pouch, a vacuum sealable pouch, a sealable formed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, ners, a box or a bag. The kit may be ized after the ories are added, thereby allowing the dual accessories in the container to be otherwise unwrapped. The kits can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat sterilization, or other sterilization s known in the art. The kit may also include other components, depending on the specific application, for example, ners, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration etc.

The present invention also encompasses a finished packaged and labeled pharmaceutical product. This article of manufacture includes the appropriate unit dosage form in an appropriate vessel or container such as a glass vial or other container that is hermetically sealed. In the case of dosage forms suitable for parenteral administration the active ingredient is sterile and suitable for administration as a particulate free solution. In other words, the invention asses both parenteral solutions and lized s, each being sterile, and the latter being suitable for reconstitution prior to injection.

Alternatively, the unit dosage form may be a solid suitable for oral, ermal, topical or mucosal delivery.

The ing examples are provided to illustrate specific instances of the practice of the present invention and are not intended to limit the scope of the invention. As will be apparent to one of ordinary skill in the art, the t invention will find application in a variety of compositions and methods.

EXAMPLES Materials and s Mice: C57BL/6J (B6) mice were purchased from Charles River. Nod/Scid/Gamma (NSG) mice were bought from the Jackson Laboratory. Sperm from the strain C57BL/6N— Nmurl‘m1‘1(KOMP)V1Cg, which contains a Nmur] deletion, was obtained from the KOMP Repository, located at the University of California Davis and Children’s al Oakland Research Institute, US. Nmurl'l' mice were generated by in vitro fertilization at the GFP 16 mice limaud Centre for the Unknown, Portugal. Ret were on a C57Bl/6J background. Mice were bred and maintained at the iMM Lisboa animal facility under specific en free conditions. Mice were systematically compared with co—housed littermate controls. Both males and females were used in this study. All animal experiments were approved by national and utional ethical committees, respectively Direcao Geral de Veterinaria and iMM Lisboa l committee. Randomization and blinding were not used unless stated otherwise. Power analysis was performed to estimate the number of experimental mice.

Analysis of gene expression microarray data: The expression profile of 239 genes d to neural pathways was performed in mouse lymphoid cells based on the Affymetrix Mouse Gene 1.0 ST Array dataset (GEO accession number GSE37448)10. Preprocessing of microarray data (including ound correction and normalization) was performed applying the robust multiarray (RMA) method30, included in the Bioconductor e afi‘y31 for the statistical software environment R32. Linear models and the B (empirical Bayes) statistic were employed in differential gene expression analysis, using Bioconductor package limma33. Plots associated with the microarray data analyses were generated in R.

Bone marrow transplantation: Bone marrow cells were flushed out from femurs and tibiae of Nmurfl' and WT littermate controls. Bone marrow cells were CD3—depleted using Dynabeads Biotin Binder (Thermo Fisher Scientific) according to the manufacturer’s instructions. 106 cells of each genotype (CD452) were injected intravenously in direct competition with a third—party WT competitor (CD45.l/CD45.2), in a 1:1 ratio, into non— lethally irradiated (150 Rad) NSG mice l). Mice were analysed at 8 weeks after transplantation.

In vitro and in vivo Neuromedin U activation: For in vitro experiments, purified lung and small intestine lamina propria ILC2s were FBS starved for 2 hours prior to ation and cultured in complete RPMI (supplemented with 10% foetal bovine serum (FBS), 1% hepes, sodium pyruvate, glutamine, streptomycin and penicillin) at 37°C. For mRNA analysis, ILC2s were stimulated overnight with recombinant mouse Neuromedin U 23 peptide (NmU23, lOOng/mL; Phoenix pharmaceuticals). Both NmU23—stimulated and l ILC2s were cultured in the presence of IL—2 and IL—7 (lOng/mL; Peprotech). ILC2s were lysed using RLT buffer (Qiagen). For cytokine protein analysis, ILC2s were incubated exclusively with din A (eBioscience) for 12 hours prior to intracellular staining. For in vivo experiments, B6 mice were injected intraperitoneal with NmU23 peptide (2ug/day) during Nippostrongylus brasiliensis infection or with a single dose of NmU23 (20pg) and analyzed after 8 hours. Control mice were treated with PBS alone.

Parasite ion: Nippostrongylus brasiliensis was maintained by monthly passages in Lewis rats as usly described". Infective (iL3) worms were kindly provided by Nicola Harris (Lausanne, Switzerland). iL3 larvae were d for 15 minutes with llin/streptomycin (300U/mL; Thermo Fisher Scientific), icin (l.5mg/mL; Sigma) and tetracyclin (30ug/mL; , washed with PBS and counted under a stereomicroscope. Mice were injected subcutaneously with 500 iL3 in 200uL of sterile PBS using a 21G needle. Mice were sacrificed at day 2 post—infection and lungs were collected and analysed.

Burden of infection: Lung te burden was quantified in finely minced lungs and as previously described". Lung were placed on sterile cheesecloth and suspended in a 50 mL tube containing PBS at 37°C for at least 4 hours. Viable worms that migrate out into the bottom of the tube were counted under a stereomicroscope (steREO Lumar V12; Zeiss).

Cell isolation: Lungs were perfused with a solution of cold PBS and 2% heparin h the right ventricle of the heart and were subsequently finely minced and ed in complete RPMI supplemented with collagenase D (0.lmg/mL; Roche) and DNase I (20U/ mL; Affymetrix) for 1h at 37°C under gentle agitation. For isolation of small intestine lamina propria cells, intestines were thoroughly rinsed with PBS, cut in lcm pieces, and shaken for 30 minutes in PBS containing 2% PBS, 1% hepes and SmM EDTA to remove intraepithelial and epithelial cells. Intestines were then digested with collagenase D (0.5mg/mL; Roche) and DNase I (20U/ mL; Affymetrix) in complete RPMI for 30 minutes at 37°C, under gentle agitation. Enteric neurons and glial cells were isolated as previously described4’35. Briefly, isolated tissues were digested with Liberase TM (7,5 ug/mL; Roche) and DNase I (20U/ mL; Affymetrix) in complete RPMI for 30 minutes at 37°C, under gentle ion. Digested organs were disrupted by passage through a lOOum cell strainer (BD Biosciences). A 40—80% l gradient centrifugation (2,400 rpm, 30 minutes at room temperature) was used for additional yte purification from lung and small intestine cell suspensions. Erythrocytes from lung, small intestine and bone marrow ations were lysed with RBC lysis buffer (eBioscience).

Flow cytometry and cell sorting: Intracellular ng was performed using IC fixation/permeabilization kit cience). Flow cytometry analysis and cell sorting were performed using BD LSRFORTESSA and BD FACSAria flow cytometers (BD Biosciences).

Data is was done using FlowJo software (Tristar). Sorted populations were >95% pure.

Cell suspensions were stained with anti—CD45 (30—Fl l), anti—TERl l9 (TER—l l9), TCRB (H57—597), anti—CD38 (eBioSOOA2), anti—CD19 (eBiolD3), anti—NKl.l (PKl36), anti—CDllc (N418), anti-Grl (RB6-8C5), anti-CDl lb ), anti-CCR6 (29-2Ll7), anti-CD127 (IL- 7ROL; A7R34), anti—0L4B7 (DATK32), anti—Flt3 (A2FlO), anti—CD25 (PC6l.5), anti—cKit (2B8), anti—Thyl.2 (53—2.l), anti—CD49b (DXS), D49a (HMOtl), anti—TCR5 (GL3), anti—Nkp46 (29Al.4), anti—CD4 (GKl.5), anti—CD31 (390), anti—IL—l3 (eBiol3A), anti—IL—4 (AABll), anti-CSF2 (MPl-22E9), anti-F4/80 (BM8), anti-chRl (MAR-l), 7AAD ity dye, Anti—Mouse CDl6/CD32 (Fc block) all from eBioscience; anti—CD80t (53—6.7), anti— KLRGl (2Fl), anti—scal (D7), anti—CCR3 (JO73E), anti—MHC—II (M5/ 1 14.15 .2) from end, anti—IL—5 (MH9A3) from BD Biosciences, anti—amphiregulin (R&D).

LIVE/DEAD e Aqua Dead Cell Stain Kit was purchased from Invitrogen. Cell tions were defined as: ILC2 — CD45+Lin'Thyl.2+KLRG1+Scal+; ILC3 — CD45+Lin' Thyl.2hiIL7R0t+RORyt+; for ILC3 subsets additional markers were employed: LTi — CCR6+Nkp46'; ILC3 NCR' — CCR6'Nkp46'; ILC3 NCR+ - CCR6'Nkp46"; NK cells - CD45+Lin'NKp46+NKl.l+CD49b+CD49a'CD127'; Lineage was composed by CD35, CDSOL, TCRB, TCRyES, CD19, Grl, CDl 1c and TERl l9; enteric glial cells — CD45'CD31'TERll9' CD49b+; T cells — CD45+CD3+ TCRB"; B cells — CD45+CDl9+; enteric neurons — CD45" CD31'TER119'RET"; phils — MHC—H-CCR3hiGR1im; Neutrophils — MHC—II‘CCR3' Gthi; Macrophages — CD3'MHC—II+F4/80+; ytes/Basophils — CD3'chRl"; Common Lymphoid Progenitor (CLP) — Lin—CD127+Flt3+ScalimcKitim; Common Helper Innate Lymphoid Progenitor (CHILP) — Lin—CD127+0t4B7+Flt3'CD25'; ILC2 precursor (ILC2P) — Lin—CD127+0t4B7+Flt3‘CD25+.

Quantitative RT-PCR: Total RNA was extracted using RNeasy micro kit (Qiagen) according to the cturer’s protocol. RNA concentration was determined using Nanodrop Spectrophotometer (Nanodrop Technologies). Quantitative real—time RT—PCR was performed as previously described". Hprt] , Gapdh and Eefla] were used as housekeeping genes. For TaqMan assays (Applied Biosystems) RNA was retro—transcribed using a High Capacity RNA—to—cDNA Kit (Applied Biosystems), followed by a pre—amplification PCR using TaqMan PreAmp Master Mix (Applied Biosystems). TaqMan Gene Expression Master Mix ed Biosystems) was used in real—time PCR. TaqMan Gene Expression Assays (Applied tems) were the following: Hprt] Mm00446968_ml; Gapdh Mm99999915_gl; Eefla] MmOl973893_gl; 115 9646_ml; 1113 4204_ml; Areg Mm01354339_ml; Il4 Mm00445259_ml; Csz Mm01290062_ml; Gata3 Mm00484683_ml; Rora MmOl l73766_ml; Nmu Mm00479868_ml; Nmur] Mm04207994_ml. ime PCR analysis was performed using StepOne ime PCR system (Applied Biosystems).

Cell signalling: Purified ILCZS from small intestine and lung were FBS starved for 2 hours before in vitro activation with NmU23 at 37°C. To test for ERK phosphorylation (Cell Signaling Technology), purified ILCZS were activated with NmU23 (100ng/mL; Phoenix pharmaceuticals) in the presence of IL—2 and IL—7 (10ng/mL; Peprotech) for 10 s prior to intracellular staining. To test ERK, calcineurin and NFAT activation, ILCZS were cultured for 1 hour with their respective inhibitor and then stimulated with NmU23 overnight before mRNA expression is. ERK inhibitor — PD98059 (Sigma); Calcineurin inhibitor — FK506 (Tocris Bioscience); NFAT inhibitor — 11R—VIVIT (Tocris Bioscience).

Calcium ing: Purified ILCZS from the small intestine were cultured with IL—2 and IL—7 (10ng/mL) and FBS deprived for 6 hours prior to calcium signaling experiments.

ILCZS were stained with Fluo—4 Direct Calcium Assay Kit (Thermo Fisher Scientific) ing to manufacturer’s protocol. Calcium (Ca2+) influx, ented by the Fluo—4 AM, was recorded over time on a BD Accuri C6 (BD Biosciences) flow cytometer as previously ed36. The recombinant mouse NmU23 was added 60 seconds after ILC2 baseline recording. Data was represented by the mean values of Ca2+ influx kinetics between the ILC2 baseline response and the peak of response after recombinant mouse NmU23 addition.

Histopathology analysis: Mice were sacrificed by cervical dislocation, and caudal lobe of the right lung was harvested, fixed in 10% l buffered formalin and processed for in embedding. Serial 4um sections were stained for hematoxylin and eosin (H&E), Luna stain, and immunohistochemistry for myeloperoxidase (MPO) was performed. Briefly, using standard protocols, antigen etrieval was performed at low pH37 in Dako PT , followed by tion with the primary antibody (polyclonal rabbit anti—human eroxidase, Dako Corp). Incubation with ENVISION kit (Peroxidase/DAB ion system, Dako Corp) was followed by Harri’s hematoxylin counterstaining (Bio Otica).

Negative control included the absence of primary antibodies. Slides were analyzed by a pathologist blinded to experimental groups and images were acquired in a Leica DM2500 cope, coupled with a Leica MC170 HD microscope camera. Quantification of inflammatory cell infiltration of the lung was performed in MPO—stained sections by manual counting of MPO—positive cells at 20x original magnification, corresponding to 0.2mm2 per field. Quantification of pulmonary eosinophils was med in Luna—stained slides by manual counting the number of granulocytes with eosinophilic granular cytoplasm in low power fields (1mm2 per field).

Microscopy: Analysis of thick gut sections ines were fixed with 4% PFA at 4°C overnight and were then included in 4% low—melting temperature agarose (Invitrogen). ns of lOOum were obtained with a Leica VTl200/VTl200 S vibratome. ns were incubated overnight or for 1—2 days respectively at 4°C using the following antibodies: mouse monoclonal anti—KLRGl (2Fl/KLRGl; Biolegend); anti—CD3 (l7A2; Biolegend).

A647 goat anti—hamster and A568 goat anti—rat were purchased from Invitrogen. After several washing steps with PBS samples were incubated with antibodies during 3 hour at room temperature and then mounted in Mowiols. Samples were acquired on a Zeiss LSM7lO confocal microscope using EC Plan—Neofluar le/0.30 M27, Plan Apochromat 20x/0.8 M27 and EC Plan—Neofluar 40x/1.30 objectives.

Statistics: Results are shown as mean i SEM. Statistical analysis was performed with GraphPad Prism software (GraphPad re, La Jolla, Calif). Student’s t—test was performed on dastic populations. Unpaired t—test was applied on samples with different variances. Results were considered significant at >"P"*P"*>"P<0.001, >""""*P<0.0001.

Example 1: Expressions of Neuromedin U Receptor 1 gNmur] ) in ILC2s Group 2 innate lymphoid cells (ILC2s) are abundant at mucosal barriers and act as key initiators of type 2 ation and tissue repair". ILC2s are activated by cell—extrinsic cytokines, including IL—25, IL—33 and thymic stromal lymphopoietinl’z. Previous reports indicated that discrete lymphocyte s and haematopoietic progenitors are controlled by dietary signals and egulators2’3’5'9 , suggesting that ILC2s may exert their function in the context of neuro—immune cell units.

To ogate whether ILC2s directly and selectively perceive neuronal—derived molecules, genome—wide transcriptional profiling of ILC2s versus their adaptive (T helper lymphocytes) and innate (ILCl and ILC3) counterparts10 was ed (Fig. . This analysis identified the gene neuromedin U receptor 1 (Nmurl ) as being ively enriched in ILC2s when compared to ILCls, ILC3s and T helper 2 cells (Figs. la—lb and Figs. 5a,5b).

This finding was med by independent quantitative sion assays in multiple subsets of immune cells, including ILCls, ILC3s, NK cells, eosinophils, mast cells, macrophages, neutrophils, dendritic cells, T cells and B cells (Fig. lc).

Nmur] encodes for a transmembrane receptor for Neuromedin U. The latter is a secreted neuropeptide found in the brain and highly expressed in the gastrointestinal tract11'14.

As such, Neuromedin U (NMU) acts as a neuronal—derived regulator in diverse physiologic processes". Neuromedin U was shown to be produced by enteric neurons, which also express the neurotrophic factor receptor RET11'13’15. In agreement, neurons in the lamina propria were main expressers of the Neuromedin U gene (Nmu), while these transcripts were not detectable in enteric neuroglia and epithelial cells (Fig. ld). rly, all analysed immune cell subsets, including dendritic cells, macrophages and B cells, had no significant Nmu expression (Fig. ld). Strikingly, reporter mice for c neurons (RetGFP)16 revealed that lamina propria CD3" KLRGl+ candidate ILC2s are adjacent to the intestinal lamina propria RetGFP neuronal network (Fig. le and Fig. 5c). Taken together these data suggest a paracrine neuron—ILC2 alk orchestrated by NMU—NMURl interactions.

Example 2: Activation Of ILC2s With Neuromedin U To explore this hypothesis, intestine and lung—derived ILC2s were purified and activated with Neuromedin U (NmU23 neuropeptide) (Figs. 2a—2f). Astonishingly, cell— autonomous activation of ILC2s with NmU23 resulted in prompt and very potent sion of the pro—inflammatory and tissue—protective type 2 cytokines genes [[5, 1113, Areg and Csz, which was paralleled by increased expression of the master type 2 transcription factor Gata3 (Figs.2a,2b). Similar finding were obtained with human ILC2 (Fig.9b,c). NmU23—dependent activation of ILC2s increased ILC2 proliferation as measured by Ki67 (Fig. 2c; Fig. 10a, 10b). NmU was shown to bind with similar affinity to two orphan class A G—protein—coupled receptors, NMURl and NMUR214.

Formal definition that NMURl activation is the molecular link between NMU— ent ILC2 activation and type cytokine production was provided by genetic ablation of Nmur] . Activation of purified ILC2s with NmU23 led to potent expression of the type 2 cytokine proteins IL—5 and IL—l3 in a NMURl—dependent manner (Fig. 2d—2f and Fig. 6a,6b).

Importantly, in viva administration of the neuropeptide NmU23 resulted in immediate and selective type 2 ne production from ILC2s, while their ve T helper cell— derived rparts were unperturbed (Fig. 2g,2h and Fig. 6c,6d). In agreement, Nmur] deficient mice had an intact ILC2 compartment, but reduced innate IL—5 and IL— 13 expression when compared to their wild—type (WT) littermate controls (Fig. 2i,2j; Fig. 6e,6f; and Fig. 7a— 7c). It was noteworthy that T helper cell—derived cytokines were unperturbed in Nmur] knockout mice (Fig. 6f).

These data indicate that the neuropeptide Neuromedin U is a potent regulator of innate type 2 inflammatory and tissue repair cytokines, via NMURl activation.

Example 3: Signalling By Activated NMURl In ILC2s To r examine how Neuromedin U controls innate type 2 responses the signalling cues provided by activated NMURl in ILC2s were investigated. In neurons activation of edin U receptors leads to increased Calcium (Ca2+) influx and ERKl/2 activation, while NFAT activity is required for type 2 cytokine production17'20. Neuromedin U—induced activation of ILC2s led to immediate and ent ERKl/2 activation, while inhibition of ERK activity upon NmU23—induced ILC2 activation resulted in impaired type 2 cytokine gene expression (Fig. 3a,3b).

Analysis of edin U—induced activation of ILC2s also led to immediate and robust Ca2+ influx, suggesting a role of the calcium dependent serine/threonine protein phosphatase eurin in NmU23—induced type 2 responses (Fig. 3c). In agreement, inhibition of Calcineurin upon NmU23 activation led to impaired innate 115, [[13 and Csz expression (Fig. 3d).

Finally, inhibition of NFAT activity upon induced NMURl activation led to similarly decreased [15, 1113 and Csz (Fig. 3e). Thus, it was concluded that the neuronal— d e Neuromedin U can operate in an ILC2—intrinsic manner by activating NMURl, which regulates innate type 2 cytokines downstream of a Ca2+/Calcineurin/NFAT e and ERKl/2 phosphorylation.

Example 4: Regulation of Mucosal Defence By ILC2 Cells To interrogate whether neuronal peptides regulate mucosal defence, how varying degrees of NMURl signals can control mucosal sion shortly after infection with the helminthic te Nippostrongylus brasiliensis21, and before adaptive T cell ses are established22 was tested. Strikingly, infection of WT mice with N. brasiliensis resulted in strongly sed Nmu expression in the lung (Fig. 4a), suggesting that Neuromedin U may regulate in viva responses to worm infection. Accordingly, administration of the neuropeptide NmU23 in N. brasiliensis infected mice resulted in very robust and immediate innate type 2 ses characterised by increased eosinophil infiltrates in the lung when compared to their vehicle (PBS) treated counterparts (Fig. .

To further explore the role of NMURl in innate type 2 responses, Nmur] deficient mice and their littermate ls were infected with N. iensis (Fig. . Strikingly, when compared to their WT littermate counterparts, Nmur] knockout mice had decreased type 2 responses, notably markedly reduced eosinophil and granulocyte rates (Fig. 4e— 4g). In line with these findings, Nmur] deficient mice had increased N. brasiliensis infection burden (Fig. 4i). Altogether, these data indicate that the neuropeptide Neuromedin U provides critical cues that te type 2 responses in viva, thus increasing ate mucosal tion against worm infections.

Example 5: Signal ation by ILC2 Cells Deciphering the mechanisms by which ILC2s perceive, integrate and respond to environmental signals is critical to understand tissue and organ homeostasis. The results reported herein establish unexpected relationships between ILC2s and their environment. A novel neuron—ILC2 cell unit orchestrated by Neuromedin U was deciphered (Fig. 8). This neuropeptide directly activates ILC2s in a NMURl dependent manner, resulting in a potent innate type 2 cytokine production downstream of ERK phosphorylation and activation of a Ca2+/Calcineurin/NFAT cascade (Fig. 8).

While it is well—established that ILC2s integrate cytokine signals, including IL—25, IL— 33 and thymic stromal lymphopoietinl’z’23 the results reported herein trate that ILC2s can more broadly integrate signals from different germ—layer—derived tissues to simultaneously regulate inflammatory and tissue repair type 2 responses and organ defence.

Thus, it is proposed that neuron—ILC2 cell units are poised to uniquely ensure potent and immediate type 2 responses in a neuromedin U—dependent manner (Fig. 8).

Previous studies demonstrated that ILC2s contribute to multiple homeostatic processes, including nutrient sensing, lism, tissue repair and ion controll’2’21’23'27.

Here it has been shown that neuromedin U is the molecular link between neuronal activity, innate type 2 responses and mucosal protection. Thus, coupling neuronal activity and ILC2— dependent immune tion may have ensured potent, efficient and integrated multi—tissue responses to environmental challenges throughout evolution. Notably, nated, neuromedin U—dependent smooth muscle contraction14 and type 2 innate immunity may have WO 09540 coevolved to control worms that have been intimate evolution partners of mammals. In line with this hypothesis, neuromedin U is highly conserved across mammalian, amphibian, avian and fish species". Finally, the t data and other independent studies indicate that the mucosal nervous system partners with ILCs and hages to ensure local tissue regulation3’4’28’29; thus it is tempting to speculate the existence of neuroimmune sensory units that regulate physiology and homeostasis at an organismic level.

Example 6: ive Expression of Nmur] and Activation of ILC2s Transcriptional analysis identified the gene neuromedin U receptor 1 (Nmurl ) as being selectively enriched in ILC2s when compared to ILCls, ILC3s and T helper 2 cells (Figs. la,lb and Figs. 5a,5b). This g was confirmed by independent quantitative expression assays in multiple subsets of immune cells, ing ILCls, ILC3 s, NK cells, eosinophils, mast cells, macrophages, neutrophils, dendritic cells, T cells and B cells (Fig. 9e). In line with this finding, activation of ILC2 with NMU23, resulted in immediate innate 115 and [[13 upregulation, while their adaptive T cell counterparts were unperturbed (Fig. 9f).

Noteworthy, after infection with Nippostrongylus brasiliensis Nmur] expression was selectively increased in ILC2 (Figs. 9a,9d). s in the lamina propria were found to be the main expressers of the Neuromedin U gene (Nmu), while these transcripts were not detectable in enteric neuroglia and lial cells (Fig. ld). Similarly, all analysed immune cell subsets, including eosinophils, dendritic cells, macrophages, B cells and T cells, had no significant Nmu expression (Fig. ld). Strikingly, reporter mice for enteric neurons (RetGFP) revealed that lamina propria CD3'KLRGl+ candidate ILC2s found at 4.7l6umi0.656 from adjacent s while their adaptive T cell counterparts are found at a significantly bigger distance (8.623 umil.447) (Fig. le; Fig. 5c; and Fig. 9g). Astonishingly, neurospherere—derived neurons stimulated with N. brasiliensis /secretory proteins (NES) rapidly up—regulated Nmu expression (Fig.9h,i), ting that s can directly sense worm products to regulate NMU production.

Example 7: Type 2 Cytokines Expressed Upon Activation of ILC2s Intestine and lung—derived ILC2s were purified and activated with Neuromedin U (NmU23 neuropeptide) (Figs. 2a—2f). Astonishingly, cell—autonomous activation of ILC2s with NmU23 resulted in prompt and very potent expression of the pro—inflammatory and tissue—protective type 2 cytokines genes [[5, 1113, Areg and Csz, which was paralleled by increased expression of the master type 2 ription factor Gata3 (Figs. 2a,2b). NmU23— ent activation of ILC2s increased ILC2 proliferation as measured by Ki67 in vitro and in viva (Fig. 2c and Figs. lOa,lOb).

Sequentially, the response of ILC2 to NMU, IL—33 and IL—25, in a dose dependent manner, was compared. Strikingly, when ed to their cytokine counterparts, IL—33 and IL—25, NMU activation of ILC2 led to a prompt, and very robust expression of innate IL—5 and IL—l3. This immediate up—regulation of NMU—induced innate type 2 cytokines was comparable to the effects observed with PMA—ionomycin activation, indicating that edin U is a uniquely potent regulator of ILC2—derived type 2 cytokines (Figs.

Example 8: Effect of NmU23—induced Cell Activation on NFAT Neuromedin U—induced activation of ILC2s led to immediate and efficient ERKl/2 activation, while inhibition of ERK activity upon NmU23—induced ILC2 activation resulted in impaired type 2 cytokine gene sion (Figs. 3a,3b). Analysis of Neuromedin U—induced activation of ILC2s also led to immediate and robust Ca2+ influx, suggesting a role of the calcium dependent serine/threonine protein phosphatase Calcineurin in NmU23—induced type 2 responses (Fig. 3c).

In agreement, inhibition of Calcineurin or its interactions with NFAT, upon NmU23 activation led to impaired innate 115, [[13 and Csz expression (Fig. 3d and Figs. lla,l lb). In agreement, NmU23—induced cell activation led to efficient translocation of NFAT from the cytoplasm to the nucleus of ILC2 (Fig. llc). Finally, inhibition of NFAT activity upon NmU23—induced NMURl activation led to similarly decreased [15, 1113 and Csz (Fig. 3e).

Thus, it was ded that the neuronal—derived peptide edin U, can operate in an ILC2—intrinsic manner by activating NMURl, which regulates innate type 2 cytokine.

Example 9: Effects of NmU23 Treatment in N. iensis Infected Mice To interrogate r al peptides regulate mucosal defence, how varying s of NMURl signals can l mucosal aggression shortly after infection with the helminthic parasite Nippostrongylus brasiliensis, and before adaptive T cell responses are WO 09540 established, was tested. Strikingly, infection of WT mice with N. brasiliensis resulted in strongly increased Nmu expression in the lung (Fig. 4a), suggesting that Neuromedin U may regulate in viva responses to worm infection. Accordingly, administration of the eptide NmU23 in N. brasiliensis infected mice resulted in a very robust and immediate innate type 2 ses characterised by increased ILC2—derived IL—S, IL—l3 and Amphiregulin, and increased eosinophil in the lung when compared to their vehicle (PBS) d counterparts (Figs. 4b—4d and Fig. 12a). Accordingly, NmU23 treatment in N. iensis infected mice led to reduced lung haemorrhage and decreased lung and intestinal parasite burden (Figs. l2b,l2f).

Example 10: Confirmation of the Role of NMURl Using Nmur] Deficient Mice To r explore the role of NMURl in innate type 2 responses, Nmur] deficient mice and their littermate controls were infected with N. iensis (Figs. 4e—4i). Strikingly, when compared to their WT mate counterparts, Nmur] knockout mice had decreased type 2 ses, notably markedly reduced ILC2—derived IL—S, IL—l3 and Amphiregulin, reduced eosinophil and granulocyte infiltrates (Figs. 4e—4g and Figs. l3a—l3c). In line with these findings, Nmur] deficient mice had increased N. iensis infection burden in the lung and ine (Fig. 4i and Fig. 13d). Altogether, these data indicate that the neuropeptide Neuromedin U provides critical cues that regulate type 2 responses in viva, thus increasing immediate mucosal protection against worm infections.

Exam le ll: Activation of lLCZ, ieada to innate t ’, e 2 e itokine ai‘odnction in viva To formally establish the link between ll..C2—aatonomous activation via NMURl and innate type 2 cytokine production in viva, bone-vinarrow (BM) mixed chimeras with NMURl sufficient and deficient BM eelis were performed. It was found that after NMU administration, Nmur] deficient ILC2 had reduced innate IL—5 and IL—l3 expression when compared to their wild—type competitive counterparts (Figs. l4a,léib)r Noteworthy, Nmur] deficient or competent T cells had unperturbed expression of these type 2 cytokines (Fig. 14c). Thus, NMU~NMUR1 operate in an ILC2—intrinaic manner to l type 2 cytokine expression in viva.

References l Cording, S., Medvedovic, J ., Aychek, T. & Eberl, G. Innate lymphoid cells in defense, immunopathology and therapy. Nat Immunol 17, 755—757 (2016). 2 Klose, C. S. & Artis, D. Innate lymphoid cells as regulators of ty, inflammation and tissue homeostasis. Nat Immunol 17, 765—774 (2016). 3 Veiga—Fernandes, H. & Mucida, D. Neuro—Immune Interactions at Barrier es.

Cell 165, 801-811 (2016). 4 Ibiza, S. et al. Glial—cell—derived neuroregulators control type 3 innate id cells and gut defence. Nature 535, 3 (2016). 5 van de Pavert, S. A. et al. Maternal retinoids control type 3 innate lymphoid cells and set the offspring immunity. Nature 508, 123—127 (2014). 6 Patel, A. et al. Differential RET ing pathways drive development of the enteric lymphoid and s systems. Sci Signal 5, ra55 (2012). 7 Veiga—Fernandes, H. et al. Tyrosine kinase receptor RET is a key regulator of Peyer's Patch organogenesis. Nature 446, 547—551 (2007). 8 Fonseca—Pereira, D. et al. The neurotrophic factor receptor RET drives opoietic stem cell survival and function. Nature 514, 98—101 (2014). 9 Veldhoen, M. & Veiga—Femandes, H. Feeding immunity: skepticism, delicacies and delights. Nat Immunol 16, 215-219 (2015). 10 Robinette, M. L. et al. riptional programs define molecular characteristics of innate lymphoid cell classes and subsets. Nat Immunol 16, 306—3 17 (2015). ll Augood, S. J characterisation of ., Keast, J. R. & Emson, P. C. Distribution and neuromedin U—like immunoreactivity in rat brain and intestine and in guinea pig ine. Regul Pept 20, 281-292 (1988). 12 Ballesta, J. et al. Occurrence and developmental pattern of neuromedin U— immunoreactive nerves in the gastrointestinal tract and brain of the rat. Neuroscience , 797-816 (1988). 13 Honzawa, M., Sudoh, T., Minamino, N., Kangawa, K. & Matsuo, H. Neuromedin U— like reactivity in rat intestine: regional distribution and immunohistochemical study. Neuropeptides 15, l—9 (1990). 14 Martinez, V. G. & O'Driscoll, L. Neuromedin U: a multifunctional neuropeptide with ropic roles. Clin Chem 61, 471—482 (2015).

Mulligan, L. M. RET revisited: expanding the oncogenic portfolio. Nat Rev Cancer 14, 173—186 (2014). 16 Hoshi, M., Batourina, E., Mendelsohn, C. & Jain, S. Novel isms of early upper and lower urinary tract patterning regulated by RetY1015 docking tyrosine in mice. Development 139, 2405—2415 (2012). 17 Hermann—Kleiter, N. & Baier, G. NFAT pulls the strings during CD4+ T helper cell effector functions. Blood 115, 997 (2010). 18 Howard, A. D. et al. Identification of receptors for neuromedin U and its role in feeding. Nature 406, 70—74 (2000). 19 z, R. et al. Identification and characterization of two neuromedin U receptors differentially expressed in peripheral tissues and the central nervous system. J Biol Chem 275, 32452—32459 (2000).

Shan, L. et al. Identification of a novel neuromedin U receptor subtype expressed in the l nervous system. J Biol Chem 275, 39482—39486 (2000). 21 Fallon, P. G. et al. Identification of an interleukin (IL)—25—dependent cell population that es IL—4, IL—5, and IL—13 at the onset of th expulsion. The l of mental medicine 203, 1105—1116 (2006). 22 Van Dyken, S. J. et al. A tissue checkpoint regulates type 2 immunity. Nat Immunol 17, 1381—1387 (2016). 23 Spencer, S. P. et al. Adaptation of innate lymphoid cells to a micronutrient deficiency promotes type 2 barrier ty. Science 343, 432—437 (2014). 24 elli, L. A. et al. Arginase l is an innate lymphoid—cell—intrinsic metabolic checkpoint controlling type 2 inflammation. Nat Immunol 17, 656-665 (2016).

Monticelli, L. A. et al. Innate lymphoid cells promote lung—tissue homeostasis after infection with influenza virus. Nat Immunol 12, 1045—1054 (2011). 26 Brestoff, J. R. et al. Group 2 innate lymphoid cells promote beiging of white adipose tissue and limit obesity. Nature 519, 242—246 (2015). 27 Nussbaum, J. C. et al. Type 2 innate lymphoid cells control eosinophil tasis.

Nature 502, 245—248 (2013). 28 Gabanyi, I. et al. Neuro—immune Interactions Drive Tissue mming in Intestinal Macrophages. Cell 164, 378—391 (2016). 29 Muller, P. A. et al. Crosstalk between muscularis macrophages and enteric neurons regulates gastrointestinal motility. Cell 158, 300—313 (2014).

Irizarry, R. A. et al. ation, normalization, and summaries of high density ucleotide array probe level data. Biostatistics 4, 249-264 (2003). 31 Gautier, L., Cope, L., Bolstad, B. M. & Irizarry, R. A. affy——analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 20, 307-315 (2004). 32 Huber, W. et al. Orchestrating high—throughput genomic analysis with Bioconductor.

Nature s 12, 115—121 (2015). 33 Ritchie, M. E. et al. limma powers differential expression analyses for RNA— sequencing and microarray studies. Nucleic Acids Res 43, e47 (2015). 34 Bouchery, T. et al. ILC2s and T cells cooperate to ensure maintenance of M2 macrophages for lung immunity against rms. Nat Commun 6, 6970 (2015).

Joseph, N. M. et al. Enteric glia are multipotent in culture but primarily form glia in the adult rodent gut. The Journal ofclinical investigation 121, 3398—3411 (2011). 36 y, T. A. et al. Lung type 2 innate lymphoid cells s cysteinyl leukotriene receptor 1, which regulates TH2 cytokine production. J Allergy Clin Immunol 132, 205—213 (2013). 37 Zhu, J. D. Myeloid cell—lineage and premylocytic—stage—specific— expression of themouse eroxidase gene is controlled at initiation as well as elongation levels of transcription. Cell Res 9, 4 (1999).

Having thus bed several s of at least one embodiment of this invention, it is to be appreciated various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention.

Accordingly, the foregoing ption and drawings are by way of example only.

Claims (64)

What is claimed is:

1. A method for increasing activity or eration of Group 2 innate lymphoid cells (ILC2s), sing contacting ILC2s with an agonist of neuromedin U receptor 1 (NMUR1) in an amount effective to se activity of the ILC2s.

2. The method of claim 1, wherein the agonist of NMUR1 is neuromedin U (NMU) or an analog thereof, or an antibody that specifically binds and activates NMUR1 or an antigenbinding fragment f.

3. The method of claim 2, wherein the NMU or analog thereof is NMU25, NMU sor protein, NMU23, or NMU8.

4. The method of any one of claims 1-3, wherein the contacting is in vitro.

5. The method of claim 4, wherein the ILC2s are contacted in an ILC2 expansion protocol.

6. The method of any one of claims 1-3, wherein the contacting is in vivo.

7. The method of claim 6, wherein the agonist of neuromedin U receptor 1 (NMUR1) is stered to a subject.

8. The method of claim 7, wherein the subject is a human.

9. The method of claim 7 or claim 8, wherein the subject is not otherwise in need of treatment with the agonist of NMUR1.

10. A method for treating a disease associated with Group 2 innate lymphoid cells (ILC2s), comprising administering to a subject in need of such treatment an agonist of neuromedin U receptor 1 (NMUR1) in an amount effective to treat the disease.

11. The method of claim 10, wherein the agonist of NMUR1 is neuromedin U (NMU) or an analog thereof, or an antibody that ically binds and activates NMUR1 or an antigenbinding fragment thereof.

12. The method of claim 11, wherein the NMU or analog thereof is NMU25, NMU precursor protein, NMU23, or NMU8.

13. The method of any one of claims 10-12, wherein the subject is a human.

14. The method of any one of claims 10-13, wherein the disease is infection, tissue , wound healing, obesity, treatable by increasing induction of type 2 immune responses, treatable by metabolic regulation, treatable by increasing eosinophils, or treatable by sing mast cells.

15. The method of any one of claims 10-14, n the subject is not otherwise in need of treatment with the agonist of NMUR1.

16. The method of any one of claims 10-15, wherein the agonist of NMUR1 is administered enously, orally, nasally, rectally or through skin absorption.

17. An agonist of neuromedin U receptor 1 (NMUR1) for use in treating a disease associated with Group 2 innate lymphoid cells ), comprising administering to a subject in need of such treatment the agonist of NMUR1 in an amount effective to treat the disease.

18. The agonist of claim 17, n the agonist of NMUR1 is neuromedin U (NMU) or an analog thereof, or an antibody that specifically binds and activates NMUR1 or an antigenbinding fragment thereof.

19. The agonist of claim 18, wherein the NMU or analog thereof is NMU25, NMU precursor protein, NMU23, or NMU8.

20. The agonist of any one of claims 17-19, wherein the subject is a human.

21. The agonist of any one of claims 17-20, wherein the disease is infection, tissue repair, wound g, obesity, treatable by increasing induction of type 2 immune responses, treatable by metabolic regulation, ble by increasing eosinophils, or treatable by increasing mast cells.

22. The agonist of any one of claims 17-21, wherein the subject is not otherwise in need of treatment with the agonist of NMUR1.

23. The agonist of any one of claims 17-22, wherein the agonist of NMUR1 is stered intravenously, orally, nasally, rectally or through skin absorption.

24. A method for treating a disease associated with Group 2 innate lymphoid cells (ILC2s), comprising administering to a subject in need of such treatment a composition comprising activated ILC2s in an amount effective to treat the disease.

25. The method of claim 24, wherein the ition further comprises an agonist of neuromedin U receptor 1 (NMUR1).

26. The method of claim 25, n the agonist of NMUR1 is edin U (NMU) or an analog thereof, or an antibody that specifically binds and activates NMUR1 or an antigenbinding nt thereof.

27. The method of claim 26, wherein the NMU or analog thereof is NMU25, NMU precursor protein, NMU23, or NMU8.

28. The method of any one of claims 24-27, wherein the subject is a human.

29. The method of any one of claims 24-28, wherein the disease is ion, tissue repair, wound healing, obesity, treatable by increasing induction of type 2 immune responses, ble by metabolic regulation, treatable by increasing eosinophils, or treatable by increasing mast cells.

30. The method of any one of claims 24-29, wherein the subject is not otherwise in need of treatment with the activated ILC2s or the agonist of NMUR1.

31. The method of any one of claims 24-30, wherein the ted ILC2s or the agonist of NMUR1 is administered intravenously, orally, nasally, rectally or through skin absorption.

32. A composition comprising activated Group 2 innate lymphoid cells ) for use in treating a disease associated with ILC2s comprising administering to a subject in need of such treatment the composition comprising activated ILC2s in an amount effective to treat the disease.

33. The composition of claim 32, wherein the composition further comprises an agonist of neuromedin U or 1 (NMUR1).

34. The composition of claim 33, wherein the agonist of NMUR1 is neuromedin U (NMU) or an analog thereof, or an antibody that specifically binds and activates NMUR1 or an antigenbinding fragment thereof.

35. The composition of claim 34, wherein the NMU or analog thereof is NMU25, NMU precursor protein, NMU23, or NMU8.

36. The composition of any one of claims 32-35, wherein the subject is a human.

37. The composition of any one of claims 32-36, wherein the disease is infection, tissue repair, wound healing, y, treatable by sing induction of type 2 immune responses, ble by metabolic regulation, treatable by increasing eosinophils, or treatable by increasing mast cells.

38. The composition of any one of claims 32-37, wherein the subject is not otherwise in need of treatment with the activated ILC2s or the t of NMUR1.

39. The ition of any one of claims 32-38, wherein the activated ILC2s or the activated ILC2s and the agonist of NMUR1 is administered enously, orally, nasally, rectally or through skin absorption.

40. A method for decreasing activity or proliferation of Group 2 innate lymphoid cells (ILC2s), comprising contacting ILC2s with an antagonist of neuromedin U receptor 1 (NMUR1) or neuromedin U (NMU) in an amount effective to decrease activity of the ILC2s.

41. The method of claim 40, wherein the antagonist of NMUR1 or NMU is an antibody that specifically binds and inhibits NMUR1 or NMU, respectively, or an antigen-binding fragment thereof.

42. The method of claim 40, wherein the nist of NMUR1 or NMU is an inhibitory nucleic acid molecule that reduces that reduces expression, transcription or translation of NMUR1 or NMU.

43. The method of claim 42, wherein the tory nucleic acid is a sRNA, shRNA, or nse nucleic acid molecule.

44. The method of any one of claims 40-43, wherein the contacting is in vitro.

45. The method of any one of claims 40-43, wherein the contacting is in vivo.

46. The method of claim 45, wherein the antagonist of NMUR1 or NMU is administered to a subject.

47. The method of claim 46, wherein the subject is a human.

48. The method of claim 46 or claim 47, wherein the subject is not otherwise in need of treatment with the antagonist of NMUR or NMU 1.

49. A method for treating a disease associated with Group 2 innate id cells (ILC2s), comprising administering to a subject in need of such treatment an antagonist of neuromedin U receptor 1 (NMUR1) or neuromedin U (NMU) in an amount effective to treat the disease.

50. The method of claim 49, wherein the antagonist of NMUR1 or NMU is an antibody that specifically binds and inhibits NMUR1 or NMU, respectively, or an n-binding fragment thereof.

51. The method of claim 49, wherein the antagonist of NMUR1 or NMU is an inhibitory nucleic acid molecule that reduces that reduces expression, transcription or translation of NMUR1 or NMU.

52. The method of claim 51, n the inhibitory nucleic acid is a sRNA, shRNA, or antisense c acid molecule.

53. The method of any one of claims 49-52, wherein the subject is a human.

54. The method of any one of claims 49-53, wherein the disease is allergy, allergic asthma, food y, eosinophilic esophagitis, atopic dermatitis, is, allergic rhinitis, allergic rhinosinusitis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, treatable by reducing type 2 immune responses, treatable by reducing eosinophils, or ble by reducing mast cells.

55. The method of any one of claims 49-54, wherein the subject is not otherwise in need of treatment with the agonist of NMUR1 or NMU.

56. The method of any one of claims 49-55, wherein the antagonist of NMUR1 is administered intravenously, orally, nasally, rectally or through skin absorption.

57. An antagonist of neuromedin U receptor 1 (NMUR1) or neuromedin U (NMU) for use in treating a disease associated with Group 2 innate id cells (ILC2s) comprising administering to a subject in need of such treatment the antagonist of NMUR1 or NMU in an amount effective to treat the disease.

58. The antagonist of claim 57, wherein the antagonist of NMUR1 or NMU is an antibody that specifically binds and inhibits NMUR1 or NMU, respectively, or an antigen-binding fragment thereof.

59. The antagonist of claim 57, wherein the antagonist of NMUR1 or NMU is an inhibitory nucleic acid molecule that reduces that reduces expression, transcription or translation of NMUR1 or NMU.

60. The antagonist of claim 59, wherein the inhibitory nucleic acid is a sRNA, shRNA, or antisense nucleic acid le.

61. The antagonist of any one of claims 57-60, wherein the subject is a human.

62. The nist of any one of claims 57-61, wherein the e is allergy, allergic , food allergy, eosinophilic esophagitis, atopic dermatitis, fibrosis, allergic is, allergic inusitis, chronic obstructive pulmonary disease (COPD), cystic fibrosis, treatable by reducing type 2 immune ses, treatable by reducing eosinophils, or treatable by reducing mast cells.

63. The antagonist of any one of claims 57-62, wherein the subject is not otherwise in need of ent with the agonist of NMUR1 or NMU.

64. The antagonist of any one of claims 57-63, wherein the antagonist of NMUR1 or NMU is administered intravenously, orally, nasally, ly or through skin absorption. WO 09540 mmmgfimm'l §\\\\ a§ (gamma; Mm 5 F73 as]: .xaw w. ..»\\\\\\\ 5: RN;H w.‘J- xxxxxx\\\\\\\\~\“------i u:ax. ‘3“am NW. \““m“\ :x“3% a.) E . A. w m .x.\\WNWvfihushikviwsw L E 3 m m. — WO 09540 H). s; mi» 3magnu. anQ3WE a 3 flu CwNu _- .m.NNX.$\.%& :waaxg , ft .NN.8 Ks N 3.; m “V;‘9 42,.aw :3. EN. NEENNN NNNNNN‘NE g E “Ua weNNNNNVNJNNNNSNNNNN.$wak . 2%,5’3’zi’3é 31.533 33$“ 2 a: fi. I35 —§}.2 {H3 SE {3.4 Cami-3 1 - figflfififi: WO 09540 E \\ a s.v. ‘5 - was‘AA: ei.Saxvmmm «M a S . anmuwE. “V A“ $4an xvm.nf Eum a. 3 SV .Li ‘ NM. 3my. RV.a. mu .Lw Mummfi m fiLiEZ \ Neuromedin U Worm V. ~?\.r infection -'~ Neuromedin U N a” “Hm§ _ Wm.. EE 9% & KXWNV.my: \: m T MWMN. . a.» we: .u m mQ“.1. Miewmmt WO 09540